New record of Neoclinus lacunicola (Actinopterygii: Perciformes: Chaenopsidae) from Ulleung Island, Korea revealed by body morphometry and mitochondrial DNA barcoding

One specimen (38.3 mm SL) of Neoclinus lacunicola Fukao, 1980, belonging to the family Chaenopsidae, was first recorded from Ulleung Island, Korea (East Sea, otherwise known as the Sea of Japan) on 5 January 2021. This species was characterized by paired external pores of incomplete lateral line running from the upper margin of the opercle, seven pairs of supraorbital cirri arranged in two rows, occipital region with a pair of cirri, and 13 rays of pectoral fin. This species is morphologically similar to the Neoclinus toshimaensis Fukao, 1980, but differs in the number of cirri on the supraorbital (6–7 versus 9–11 cirri). This study documents the first report of N. lacunicola in Korean waters and proposes the new Korean name of ‘eol-lug-bi-neul-be-do-la-chi’ for the species. For the confirmation of the identity of the species, a partial gene sequence of the mt COI (570 bp) of N. lacunicola was obtained for the first time.


Introduction
The family Chaenopsidae comprises 96 species belonging to 14 genera distributed worldwide, with one genus and eight species occurring in Japanese waters (Aizawa and Doiuchi 2013;Nelson et al. 2016), but only two species representing a single genus have recently been recorded in Korean waters (Kim and Kang 1991;Myoung et al. 2021). The tube blennies (Chaenopsidae) are reef-associated marine fishes, mainly distributed in warm North and South American waters (Nelson et al. 2016). Generally, small-sized, usually elongated and compressed, with a large mouth. The supraorbital and nasal cirri are present or absent (Murase et al. 2015). They exhibit a hiding behavior in crevices between rocky matrices, but occasionally inhabit empty gastropod shells (empty barnacles and empty worm shells) (Stephens and Springer 1971;Fukao 1980;Fukao and Okazaki 1987). Such behavior is advantageous to protect their bodies from potential predators and to care for their spawned eggs within the habitats (Murase and Sunobe 2011; Froese and Pauly 2020).
Neoclinus lacunicola Fukao, 1980 was firstly reported as a new species in 1980 (Fukao 1980) and later, several studies have reported on the taxonomic checklists, phylogeny, and encyclopedic information (Patzner et al. 2009;Aizawa and Doiuchi 2013;Lin and Hastings 2013;Soniyama et al. 2020). However, there is still a lack of information on its biology and ecology.
To date, one species of tube blennies, i.e., Neoclinus bryope (Jordan et Snyder, 1902) has firstly been reported in Korean waters (Kim and Kang 1991) and, more recently, Myoung et al. (2021) documented the first record of Neoclinus chihiroe Fukao, 1987 in Dokdo, Korea; consequently, a total of two species have been recorded in Korean waters. This study provides the first record of an additional chaenopsid species (N. lacunicola) collected from Ulleung Island, describes its morphological characteristics, and records its mitochondrial DNA cytochrome c oxidase subunit I (mt COI) sequences.

Methods
One specimen of N. lacunicola was collected from Tonggumi (37°27′33.50′′N, 130°51′28.84′′E) in the coastal waters of Ulleung Island, Gyeongsangbuk-do, Korea (East Sea, otherwise known as the Sea of Japan), by a SCUBA diver on 5 January 2021 (Fig. 1). Sampling was conducted at a depth of approximately 22 m, close to the shoreline during the day and the sample was transported to the laboratory immediately after capture in a living condition, using a small fishbowl with seawater. In the laboratory, an image of the specimen was taken, it was euthanized and then meristic counts and body morphometrics were recorded following the method of Hubbs and Lagler (1958). The specimen was measured to the nearest 0.1 mm using digital Vernier calipers and a microscope. The specimen was then preserved in 5% formalin for 24 h and later transferred to 70% ethanol for depositing at the Marine Biodiversity Institute of Korea (MABIK).
To compare molecular data, total genomic DNA was extracted from the muscle tissue using 10% Chelex resin (Bio-Rad, Hercules, CA). A portion of the mitochondrial COI gene was amplified using universal primers (Ward et al. 2005). PCR was performed in a 20 µL reaction tube containing 1 µL genomic DNA, 2 µL 10× PCR buffer, 2 µL 2.5 mM dNTP, 1 µL of each primer, 0.1 µL Ex-Taq DNA polymerase, and 12.9 µL sterile distilled H 2 O, using a thermal cycler (MJmini PTC-1148, Bio-Rad USA). The PCR profile consisted of initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 1 min, annealing at 50°C, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. PCR products were purified using ExoSAP-IT (United States Biochemical Corporation USA) and sequenced, using an ABI PRISM BigDye Terminator v.3.1 Ready Reaction cycle sequencing kit (Applied Biosystems Inc. USA) on an ABI 3730xl DNA analyzer (Applied Biosystems Inc.). We compared our molecular data with those of the mtDNA COI sequences from other Neoclinus species obtained from Gen-Bank (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov). Sequences were aligned using ClustalW (Thompson et al. 1994) in BioEdit, version 7 (Hall 1999). The genetic divergences were calculated using the Kimura 2-parameter (K2P) (Kimura 1980) model with Mega 6 (Tamura et al. 2013). Phylogenetic trees were constructed using the neighbor-joining method (Saitou and Nei 1987) in Mega 6 (Tamura et al. 2013), with confidence assessed, based on 1000 bootstrap replications.
Description. The body counts, measurements, and proportions of body parts are shown in Table 1. Body elongated and compressed (Fig. 2). Head short and round. Snout short, but mouth relatively large; posterior tip of upper jaw beyond posterior margin of eyes. Distance of interorbital area narrow. Single nasal cirrus on posterior rim of nostril; cirrus long, thin, and slender with four branches. Seven pairs of supraorbital cirri arranged in double rows with four cirri on outer row and three cirri on inner row and thick and short branches on each cirrus. Occipital region with single pair of cirri; each cirrus short, wide, and monotonous (Fig. 2B). Specimen having single dorsal fin and dorsal fin membrane shallow notched; soft part of ray usually higher than spinous part. Origin of dorsal fin located forward to origin of pelvic fin. Origin of soft part of dorsal fin ray located behind anus. Caudal fin separated from both dorsal and anal fins. End of caudal fin rounded. Entire margin of anal fin serrated; origin of anal fin located in front part of central portion of body. Pectoral fin rounded and located behind pelvic fins.   Head red and body uniformly white with black patterns (Fig. 2). Upper part of head red and white spots scattered under eyes and on chin. Red part of head appearing before fourth spine of dorsal fin and color gradually becoming faded to fourth spine of dorsal fin. Jaw and pelvic fins light yellow. Cirri in nostrils almost transparent. Lower part of supraorbital cirri red, but tip of cirri transparent. Background color of body white, with eight large black bands going down from dorsal to lower middle body and starting points of eight black patterns irregularly appear under the basement of dorsal fin. Black blotch surrounded by red be-tween second and third dorsal spines. Spine and soft rays of dorsal fin having irregular red color and transparent membrane. Upper part of opercular membrane having no black blotch. Overall, pectoral fin is pale red. Basement of pectoral fins with no black dot. Pelvic fins covered in yellow. Anal and caudal fins light red but almost transparent.
Based on an analysis of the mitochondrial cytochrome oxidase subunit I gene (COI) sequence (570 bp), the presently reported specimen was different from other six species of the genus Neoclinus with genetic distances of 0.141-0.220 (Fig. 3).

Discussion
In this study, one specimen of chaenopsid fishes has been collected from Ulleung Island with body elongated and compressed, no scales, supraorbital and nasal cirri present (Williams 2003;Nelson et al. 2016). The body morphometrics of the specimen were well matched with the original description of N. lacunicola with paired external pores of the incomplete lateral line, seven pairs of supraorbital cirri, occipital region with a pair of cirri, and 13 rays of pectoral fin (Fukao 1980;Aizawa and Doiuchi 2013). There are some differences between the presently reported specimen and that described by Fukao (1980); the differences were mainly the anal origin to pelvic insertion and slightly shorter length of caudal peduncle and longest pectoral ray length. Nonetheless, it is consistent with original descriptions with the main features of no black spot between first and second dorsal spines, occipital region with a pair of cirri, and seven pairs of supraorbital cirri (Fukao 1980;Aizawa and Doiuchi 2013;Murase et al. 2015). The body morphology of N. lacunicola is almost similar to Neoclinus toshimaensis Fukao, 1980, but distinctly different in the number of supraorbital cirri (6~7 in N. lacunicola vs. 9~11 in N. toshimaensis) (Fukao 1980;Aizawa 2002;Aizawa and Doiuchi 2013). The findings of two chaenopsids of the genus Neoclinus, namely, N. bryope and N. chihiroe were recently reported from Korean waters (MABIK 2020;Myoung et al. 2021). Neoclinus lacunicola is easily distinguished from the other two by lacking the black spot between first and second dorsal spines (without a black spot in N. lacunicola vs. with black spot in N. bryope and N. chihiroe) (Fig. 2B) (Aizawa 2002;Aizawa and Doiuchi 2013). The specimen collected in this survey had a black spot in the membrane of the second and third dorsal spines. This morphological character i.e., the presence or absence of a black spot between first and second dorsal spines, is one of the things that distinguish between males and females of Neoclinus monogrammus Murase, Aizawa et Sunobe, 2010 (see Murase et al. 2010) and it cannot be classified as Neoclinus species according to the taxonomic key suggested by Aizawa and Doiuchi (2013). For the presently reported specimen, yellow pelvic fins are key to distinguishing it as a female, despite the presence of black spots in the second and third dorsal spines membrane (Murase et al. 2015). Therefore, the presence or absence of the black spot is thought to be more variable in the same species than previously considered. In addition, our specimen 5.0 5.7 6.1-6.9 6.3-7.1 Length of 1 st dorsal-fin spine 5.5 6.4 5.9 7.0 Longest dorsal spine length 9.0 (13 th ) 8.4 (13 th ) --Longest dorsal soft ray length 11.8 (11 th ) 10.2 (11 th ) --Longest anal ray length 9.1 (24 th ) 8.2 (24 th ) --Length of last dorsal-fin spine 8.1 -6.5-8.2 7.7 Length of 1 st dorsal-fin soft ray 9.7 -8.7-10.5 9.3 Length of 1 st anal-fin spine 5.0 3.9 2.6-4.4 4.0 Length of 1 st anal-fin soft ray 7.6 6.4 5.7-6.9 7.9 Longest pectoral ray length 16.4 (9 th ) 12.5 (9 th ) --Longest pelvic ray length 9.4 (2 nd ) 9.1 (2 nd ) --was well distinguished from Neoclinus nudus Stephens et Springer, 1971 and N. bryope was well separated from other congeners by genetic distances (d) of range 0.141 and 0.191 when a comparison of mitochondrial DNA COI sequences is made (Fig. 3). Therefore, this study documents the first record of N. lacunicola in Korean waters and suggests the new Korean name of 'eol-lug-bineul-be-do-la-chi' for the species. Chaenopsid fishes, occurring in Korean waters, have previously been known only as two species (N. bryope and N. chihiroe), but an additional species has now been reported through this study. The ecological characteristics of chaenopsid fishes are known that they do not have large movements and inhabit small caves between rocks or in empty gastropod tubes (Stephens and Springer 1971;Fukao and Okazaki 1987;Fukao 1990). Due to this characteristic, their distribution ranges are expected to be considerably narrow (Hongjamrassilp et al. 2020). However, although the mobile abilities are limited within the habitat ranges, their larvae have potential dispersal possibilities through the oceanographic processes, including currents (Murase et al. 2015). According to Murase et al. (2015), the distribution range of N. lacunicola has been extended northwards along with the Tsushima Current and this study confirmed such northern distribution of the species. Although this species has firstly been reported to Korean waters, it is also expected that a number of individuals live in eastern Korean waters as well as Dokdo. In addition, because of the scarcity and the narrow habitat range of the species, there is a need for more ecological information, including their genetic group structure, geographic distribution range, and larval dispersals.

Introduction
The most recent inventory of fish species from Réunion includes 984 marine and freshwater species belonging to 164 families (Fricke et al. 2009). This inventory conceals an unequal sampling effort between habitats because the majority of censuses were carried out on the coral reefs of the western side of the island (Pinault et al. 2013). Recent studies have provided a better understanding of the sandy and alluvial zones of the northern half of the island, the volcanic zone of Piton de la Fournaise, in the southeast, and the coastal habitats of an artificial reef or wrecks (Pinault 2013;Pinault et al. 2014). Throughout these studies, many new species were discovered and added to the island's faunistic inventory, and there are likely many more species yet to be observed.
As a part of the shark risk reduction, the missions carried out by the Shark Security Center, underwater cameras were deployed on the west and south-west coast of the island, primarily in sandy and detrital areas. The objective of these cameras was to record the frequentation of these sites by potentially dangerous sharks, as well as obtain a record of fish species living in these areas.
develop discontinuously over 25 km (Tessier et al. 2008). The southern and eastern regions, exposed to wind and swell, are sparsely inhabited. They are marked by the volcanic activity of Piton de la Fournaise and are characterized by recent flows and high coastal cliffs. Finally, in the northern region, also very urbanized, stretch pebble shores and alluvial black sand beaches.
As part of the monitoring and surveillance of shark populations in Réunion, Baited Remote Underwater Video Systems (BRUVS) were deployed all around the island. While sharks were the target species, an exhaustive list of all observed fish species was compiled.
Additionally, a single, freshly caught specimen of the genus Torquigener was brought to the research team for identification. This specimen was used to collect all morphometric information, measuring all characteristics outlined in Sabour et al. (2014) using calipers.

Results
Out of a total of 265 deployments of BRUVS, Torquigener flavimaculosus Hardy et Randall, 1983 was observed 22 times at depths ranging from 7 to 50 m ( Fig. 1), but the majority of observations were made between 7 and 18 m on volcanic sand substrates. These fish often occurred in small groups ranging from 2 to 11 adult individuals.
Torquigener flavimaculosus is distinguished by the following set of characters which we used to formally identify the species (Fig. 2): the body is elongated. The  eye is surrounded by the dorsal lateral line. The lower margin of the eye is above the level of the corner of the mouth and above the top of the pectoral fin base. The mouth terminal is at the level of the upper end of the pectoral fin. The chin is distinct. The dorsal and anal fins are elongated and pointed. Small spinules are present on the belly, head, sides, and back in a spot that does not reach the dorsal fin. The lower border gill has a cartilaginous spur. The posterior margin of the gills covers small irregularly distributed spines. The dorsal body surface is brown with gray-whitish spots. A median lateral line of yellow-orange spots is distinguished, followed by a pale yellow area, separating the dorsal surface from the white ventral surface. Vertical yellow-brown stripes on the cheek are separated by irregular white stripes. The caudal fin shows brown spots. The dorsal fin is lightly spotted with white. The anal and pectoral fins are transparent. Table 1 summarizes the main morphometric and meristic data of the captured specimen. They are consistent with the description of this species provided by Hardy and Randall (1983) and Golani (1987) for this species which is referenced in the classification as being part of the Class of Actinopterygii, of the Order of Tetraodontiformes, of the Family of Tetraodontidae, of the Genus Torquigener Whitley, 1930 and of the species Torquigener flavimaculosus.

Discussion and conclusion
The yellowspotted puffer, Torquigener flavimaculosus, is found in the western Indian Ocean from the northern Red Sea to Kenya and in the Arabian Gulf and Seychelles (Froese and Pauly 2021). It has also been recorded in the Mediterranean Sea, as a Lessepsian migrant from the Red Sea, by Golani (1987) in Haifa Bay, Israel. Later, the species has also been recorded from Turkish coastal waters, the north-eastern Mediterranean Sea (Bilecenoglu 2003(Bilecenoglu , 2005; Erguden and Gurlek 2010), and from Greek waters (Corsini-Foka et. al. 2006;Zenetos et al. 2008). By comparing the morphometric and meristic characters of our specimen with other Mediterranean individuals, we do not notice any striking differences in their proportions. The specimen was initially identified visually using body markings and coloration, these matching morphometric measurements help confirm the identification as Torquigener flavimaculosus. This is the first sighting of this species in Réunion, where it has been recorded on several occasions, close to the substrate, on a black sandy bottom, between 7 and 50 m of depth. This particular habitat is characterized by an endogenous fauna of mollusks, worms, and echinoderms, which represent abundant food for this invertivorous species (Froese and Pauly 2021). The associated ichthyological fauna is relatively poor and only a few small pelagic such as Selar crumenophthalmus (Bloch, 1793) or Decapterus macarellus (Cuvier, 1833) occasionally congregate there (Turquet et al. 1998). The presently reported T. flavimaculosus represents a genus with 21 species (Froese and Pauly 2021), none of which have yet been recorded in Réunion. The validation of this species in the area adds to its distribution area and completes the ichthyological inventory of Réunion.
Very little is known about the reproduction of fishes of the genus Torquigener. At least one species is known to fabricate intricate nests to attract potential mates (Kawase et al. 2013). Males were observed defending the nest after reproduction but left once the eggs have hatched (Kawase et al. 2013). It is unknown whether the hatchlings stay demersal or whether they become pelagic and part of zooplankton. According to Froese and Pauly (2021), another species, Torquigener hypselogeneion (Bleeker, 1852), is present in Seychelles; it would be interesting to know if this species is present in Réunion. Pelagic larval dispersal is known to occur between the Seychelles and Réunion Island, which could have been a way for the species to colonize the coastal waters of Réunion (Crochelet et al. 2016).

Introduction
Given the increased use of modern fishing devices, many marine and freshwater fish resources are intensively exploited, resulting in rapid declines in some fish stock, affecting national economies, local communities' socio-economic well-being, and even their protein saecurity (Tang and Chen 2004;Branch et al. 2011). Subsequently, as a frequently used tool to control fishing effort and protect feeding and spawning areas, time-area fishing closure measure has been taken into account by governments of many countries. The effect of this measure has been assessed on fishing strategies regarding the fishing mode, variations in the population structure of a particular fish, incidental megafauna catches, and social well-being impacts (Britton 2014;Escalle et al. 2016;O'Farrell et al. 2016), but more rarely, on overall fish stock.
With the improvement of instrument performance and the development of computer software, the hydroacoustic method has increasingly become one of the main methods for fishery stock assessments due to its advantage of a rapid, economic and extensive coverage of the water cross-section at distances large enough not to affect fish behavior (Simmonds and MacLennan 2005), and now has been widely used in many bodies of water (Hale et al. 2008;Pavlov et al. 2010;Gerasimov et al. 2019;Guo et al. 2019).
In China, overfishing has also become one of the most serious problems in inland waters, as well as in oceans, and time-area fishing closure measures have been implemented since 2002. However, there has been no published assessment of overall fish stock following a closure in any rivers. Qiantang River is the largest river in Zhejiang province with a basin area of about 55 000 km 2 . Except for drinking, it also has functions of power generation, flood control, irrigation, and tourism. The river was historically rich in fish resources, and fish harvests date back several centuries, the highest take was recorded in 1960 with 5318.2 tones. Although harvest strategies incorporating seasonal bans and restricted fishing grounds have been conducted several years before 2019, which were mainly conducted in different tributaries of the river, the effects were not ideal due to inconsistency. The year 2019 was the first year that Qiantang River was included in the unified fishing ban system for the south of Yangtze River basin. Subsequently, all captures except recreational fishing should be banned in the main channel of Qiantang River from 1 March through 30 June every year in the future. So, what happened to fish stock after a four-month fishing closure in 2019?
Here, based on the data obtained from field fish collections and hydroacoustic surveys before and after the implementation of fishing closure in the main channel of Qiantang River, we compared the distribution of species in the research area, identified the dominant species, and evaluated changes in the fish size, density, biomass, and distribution. Our findings will present a description of characteristics of the temporal change effected by the closure and provide a scientific basis for the protection of fish stock in the river.

Research area
The study area belongs to a section of the main channel of Qiantang River, an important fishing stretch of water for fishermen in Fuyang and Tonglu counties (Fig. 1). This area is located between 29°52.6′N and 30°3.4′N, and 119°45.8′E and 119°59.5′E with a length of 29.9 km from east to west, with an average water depth of 14 m, a water volume of 0.31 km 3 , and a maximum water depth of 35 m.

Field collection
Two fish removals were conducted by the hired fishermen in the research area in July 2018 and July 2019. The research area was evenly divided into 4 fish sampling sections, and the fishes were caught by deploying ground bamboo cages and multipanel nylon gillnets with mesh sizes ranging from 1 to 8 cm in each sampling section. All fishes caught were humanely euthanized, counted, and identified. Each specimen was measured to the nearest 0.1 cm (total length, L) and weighed to the nearest 0.01 g (weight, W) simultaneously. where %N is the individual number of a certain species relative to the total number of fishes, %W is the weight of a certain species relative to the total weight of all fishes, and %FO is the frequency of occurrence of a certain species among the 4 fish sampling sections. According to the evaluation criterion of Zhang et al. (2016), the species is considered to be dominant when its IRI value is above 2000. For the most dominant fish species, the length-weight function, where W is the weight [g], L is the total length [cm], a is the intercept and b is the slope, was fitted with a simple linear regression model using log-transformed data.

Acoustic surveys
Two hydroacoustic surveys were conducted on 17 July 2018 and 19 July 2019 by using a BioSonics split-beam DT-X echosounder (BioSonics, Seattle, WA, USA) with a 201 kHz transducer, an integrated GPS (Garmin 17xHVS, Garmin Ltd., Olathe, KS, USA) and a computer. Surveys were conducted during daylight sunshine, and the daily mean value of water temperature was 26.3°C and 23.5°C, respectively. A fishing boat was hired to help surveys at a speed of 2.0-2.5 m/s in a zig-zag route. During surveys, the transducer face was held 40-50 cm below the water surface. A standard BioSonices 36 mm tungsten carbide sphere was used to calibrate the transducer before each survey (Foote et al. 1987). The degree of coverage (D) was calculated for each survey according to the formula of Aglen (1983). Here, the degree of coverage was 6.14 and 6.36, respectively, which were both higher than 2, indicating the sampling error of density estimates to be less than 10% (Godlewska et al. 2009). The usage method of instruments was consistent with the scheme of Guo et al. (2019).

Acoustic data analysis
The hydroacoustic data were analyzed using BioSonics Visual Analyzer software 4.1 (BioSonics, Seattle, WA, USA), consistent with the scheme of Guo et al. (2019). The fish tracking parameters were set to values in Table  1. Only data between 1.5 m of the transducer and 0.5 m off the bottom were used in order to exclude dead zones. Fish density estimates were calculated by echo integration, defined as the summation of the volume backscattering strength (Sv) divided by the backscattering cross-section (σ bs ), derived from the mean echo intensity (target strength, TS) of individual fish. With set appropriate threshold (-60 dB) and manual corrected, data for each transect were cleared of noise (Zhou et al. 2016). The integration interval was set to 1200 pings.

Calculation of fish size and biomass
Based on single echo detections (SED), the TS distributions were examined. The received echo signals were compensated depending on their range (R)[m] by the time-varied gain (TVG). A 40 log(R) TVG was applied to measurements of TS, whereas a 20 log(R) TVG was used to the measurements of Sv used when echo integration. Then, the mean value of TS (based on SED from -60 dB to -30 dB in 2 dB bins) was converted to mean length of tracked fish using the empirical formulas for TS-length relation (Foote 1998); mean length was converted to weight using the calculated length-weight function, W = aL b , for the most dominant fish species; the weight was multiplied by the total density of transects; and the mean weight of the transects was then calculated as the mean biomass.

Statistical data analysis
Statistical analyses were performed using SPSS 16.0 and Excel 2007. Data are presented as mean ± standard error of the mean (SE). Prior to analysis, all data were tested for homogeneity (Levene's test). The nonparametric Kruskal-Wallis H test or one-way ANOVA were used to analyze the effects of fishing closure on mean TS, length, density, and biomass. To compare differences in fish density, water depths were divided into five intervals: 0-5 m, 5-10 m, 10-15 m, 15-20 m, 20-25 m, and >25 m. A two-way ANOVA was also performed to test the effects of water depth and fishing closure on fish densities. All tests were considered significant at a probability level of P < 0.05 (95% confidence). Box plot was performed in R 3.6.1 environment (R Development Core Team 2010).

Fish biodiversity resulting from traditional capture method
In total, 47 and 44 fish species were identified before and after fishing closure respectively, both representing
Echoes of more than -58 dB target strength were considered fish. The TS before and after the fishing ban both ranged from -58 dB to -34 dB. To compare differences in fish population sizes, fishes were divided into small-, mid-, and large-sized categories, based on their length (TS conversion). TS distributions varied between two surveys. Before closure, mid-sized fish of TS -50 to -38 dB (≈11−48 cm) accounted for 59.81% of the population, while smallsized fish under -50 dB accounted for 38.51% of all fish (Fig. 2). This was a little different from the results obtained after closure, of which mid-sized fish accounted for 58.86%, while small-sized fish 40.24% (Table 2).

Relation between water depth and fish density
More than 50% of fish species were found in the >20 m category before closure. While fish gradually moved towards the upper water layer, leading to the most distributed  layers of fish being located at 15-20 m, 5-10 m, and 10-15 m depth after closure, accounting for 24.95%, 20.44%, and 18.30% of the total fish in the research area, indicating a wider spread of fish (Table 3). However, the arithmetic means of density was not significantly affected by water depth (F 5, 1850 = 1.855, P > 0.05), but affected by the fishing closure (F 1, 1850 = 6.571, P < 0.05). As showed in Fig. 3, the medians of all transects in different depth categories after closure were all larger than those before closure, indicating a higher mean fish density after closure. Meanwhile, fewer outliers were found in six different depth categories after closure than before, indicating a more discrete density distribution of the fish before closure.

Effects of time-area closure on fish stock
With a time-area closure, an area of water is closed to fishing with particular fishing gears during certain time periods. Originally, a time-area closure is commonly focusing on a single, target species and a single fishery (e.g., parrotfish, shrimp, tropical tuna) (Armsworth et al. 2011;O'Farrell et al. 2016), and now used to manage spatially and temporally acute bycatch problems (Goodyear 1999

The necessity of a time-area fishing closure in Qiantang River
In recent years in Qiantang River, with the socio-economic development, protection of fish stock has become increasingly serious. Although modern gears in the river are limited to gillnets, which are three-tiered with mesh sizes varying from 1 to 8 cm, the fishing mode results in indiscriminate harvesting of undersized and non-tar-  get species. The volume of fish stock in the river has declined, and the fish population structure is known to have changed. According to literature records, 136 fish species could be collected in this river in the late 1970s, receding to 122 species in 2018. Also, it has been found that some native fishes, e.g., Acipenser sinensis Gray, 1835, Psephurus gladius (Martens, 1862), Tenualosa reevesii (Richardson, 1846), and Protosalanx hyalocranius (Abbott, 1901), have disappeared and some alien ones, e.g., Lepomis macrochirus Rafinesque, 1819 and Ictalurus punctatus (Rafinesque, 1818), have become common. Thus, a fishing closure is conducive to the sustainable development of fishery in the river.

Effects of the fishing closure on fish biodiversity
In practice, the general closure period in Qiantang River included in the unified fishing ban system for the south of Yangtze River basin is from 1 March through 31 June, lasting for 4 months. It is confirmed that many economically important fish propagate at this time every year, and the aggregating behavior of breeding fish makes them more vulnerable to capture. Ley et al. (2002) found high diversity and productivity in tropical mangrove-dominated estuaries after closure. Here, the decline of three species after closure may be related to the contingency of fishing. Marks et al. (2015) also confirmed that total catch rates after the closure were significantly higher than before, differences in the size composition of species reflected both the increased survival of older fishes and higher recruitment success. Furthermore, we concluded that the conservation effect would be different for different fish. After a four-month fishing closure, the most dominant species remain unchanged. By contrast, the subdominant and common species changed to some extent. Different from small fishes (Distoechodon tumirostris, Carassius auratus, and Squalidus argentatus) contributing to subdominant species before closure, migratory species (Coilia nasus) and releasing species (Hypophthalmichthys molitrix and Cyprinus carpio) became the subdominant species after closure. Meanwhile, in addition to Tachysurus nitidus and Megalobrama terminalis remaining unchanged, the other two fishes of the first four common species changed. Thus, it seems that the presently reported fishing ban system could help migratory fish to propagate quickly, rather than being caught when spawning. At the same time, the fishing ban also led to a significant decrease of capture amount for releasing species during the closed period, resulting in a significant increase of abundance in the river.

Effects of the fishing closure on fish size, density, and biomass
It is accepted that such a closure can effectively protect fish spawning and hatching, and is also beneficial for the growth of fish larvae, to promote the natural supplement of fish resources and the self-restoration of the ecological environment in the waters (Tang and Chen 2004). Our results also confirmed this view by using the hydroacoustic method, which showed that the mean fish density and biomass after the closure were both significantly greater than that before it, while the derived mean length after the closure was lower than before. In detail, the mean length of fish decreased after the time-area ban, i.e., the proportions of small-sized fish increased (40.24% vs. 38.51%), while mid-sized (58.86% vs. 59.81%) and large-sized fish (0.90% vs. 1.68%) decreased in July 2019. The mean weight also showed the same trend. Reproduction protection during the closure could somewhat explain the reason. The 2019 fishing closure increased the survival of older fishes and most juvenile fishes, made them supplementary to the total population, increasing fish abundance and the small size composition of fish species.

Effects of the fishing closure on fish distribution
Fishing closure resulted in a change of fish distribution. It is accepted that time-area closures are often preferred for more mobile pelagic species (Hobday and Hartmann 2006;Grantham et al. 2008;Game et al. 2009), maybe due to their easy capture by gillnets with relatively fixed mesh size. In this study, a reduction of these pelagic fish stock, a more discrete size of uncaught fish, and a distribution in deeper water were disclosed before closure. For example, more than 50% of fish were concentrated in >20 m layer. However, after a four-month fishing closure, there was no significant difference in the distribution of water depth, with more than 50% of the fish distributed in 5-20 m layer. Thus, it seems that the fishing closure of 2019 protected the pelagic fish and made fish more evenly distributed.

Conclusion
The reasons for the time-area ban policy are biological, concerned with protecting and restoring fish stock, not only impacting catches of target species, but also non-target species that can be sold, and non-target species that do not have commercial value for fishermen (Chumchuen et al. 2016). Since 2019, a four-month fishing closure from 1 March through 31 June has been adopted in the main channel of Qiantang River of China every year, which has a great positive effect on fish size, density, and biomass, and makes fish more evenly distributed. Future continuous multi-year monitoring combining the traditional capture and hydroacoustic survey methods needs to be carried out, and more additional closure time may be needed except for the critical life stage of important endangered fish species, to realize the sustainable development of fish in the river.

Introduction
In the recent past, the production of ornamental fish feed was mostly dependent on bycatch and other aquatic organisms being used as live feeds. However, due to the increasing pressure on natural resources, organized industrial sites have been established for the breeding and marketing of ornamental fishes. Nowadays, feed and aquarium equipment used in ornamental fish farming have emerged as an industry that appeals to the global market. In developed and developing countries, the ornamental fish-farming sector has developed with major commercial importance within the aquaculture industry (Sales and Janssens 2003; Hekimoğlu 2006; Dominguez and Botella 2014).
Nutrition is one of the most important elements for the sustainability of fish farming. In addition to carbohydrates, proteins, and fats in the structure of the feed used in nutrition, minerals, vitamins, and anabolic steroids are also used as additives.
Anabolic steroids contain androgen (male sex hormone) and estrogen (female sex hormone), which are commonly used as growth supplements in feed. Steroid hormones can be used in feed without any biological loss since they are resistant to catabolism by digestive enzymes. Thus, fish can be supplemented with hormones without incurring any handling stress compared to other invasive methods like injection, etc. Moreover, administering hormones to multiple fish individuals is a simple procedure (Hoşsu et al. 2003).
The wide use of growth promoters in animal husbandry using the anabolic sex steroids has attracted their application in the aquaculture industry to shorten the production cycle and lower the production cost (Gannam and Lovell 1991).
Anabolic agents (or anabolics) have been defined as substances that enhance the protein synthesis of livestock by positively affecting the nitrogen balance. Therefore, anabolic hormones enable lean meat production and rapid weight gain of fish and livestock with less feed by expediting muscle development. Most of these are steroidal male and female sex hormones (both natural and synthetic) and non-steroidal substances with anabolic effects. Furthermore, growth hormones, somatomedins, insulin, and thyroid gland hormones have also been documented to affect the growth rate positively (Mercure et al. 2001;Karabulut 2008).
Sex reversal by hormone treatment and its application in certain expensive aquarium fish, especially for which market value depends on the sex, have drawn the attention of many ornamental fish farmers and this situation has demanded the requirement of more studies on this subject. Studies related to color enhancement in ornamental fish farming have gained momentum in recent years and particularly the use of anabolic steroids is of great interest. Apart from sex reversal, steroids are also used for enhancing the growth or color of fish for visual appeal.
Cichlids, being one of the most visually appealing fishes in the industry, are amongst the aquarium fish, for which market value depends on sex. The emperor red cichlid, Aulonocara nyassae Regan, 1922, attracts attention, especially due to its color in shades of red. The colors of the males are much brighter red with a noticeable difference compared to the females and their dorsal and anal fins are more pointed. Females also usually have grey and dark tones. In this study, the emperor red cichlid was used due to its importance in the aquarium industry and there is a lack of information on the effect of sex steroids on the growth and coloration of this species.

Materials and methods
Experimental design. A 60-day study was conducted in a closed system, where the juveniles of emperor red cichlid (A. nyassae) were acclimatized with the control/basal diet for 15 days. After which, 15 fish with a similar shade of color and about 5 months old (unsexed) were weighed individually (0.71 ± 0.01 g) and placed into each aquarium (30 L) in five different groups, in triplicate. Five different groups consisted of control (without hormone), 50 mg · kg -1 17α-MT, 100 mg · kg -1 17α-MT, 50 mg · kg -1 17β-Es (E 2 ), and 100 mg · kg -1 17β-Es (E 2 ). The basal feed had the approximate composition of 46.5% crude protein, 8.2% crude fat, 3.3% crude fiber, 5.0% moisture, and 5.3% crude ash. The fish were fed twice a day (10:00 h, 17:00 h) for 60 days a diet till satiation. Experimental fish were weighed at the beginning of the experiment and the last day of the experiment (on the 60 th day). Waste materials were siphoned out every day and replacement water was added. In each experimental treatment group, fish were acclimatized to aquarium and feeding conditions for a week. The temperature of the aquarium unit was set to have a water temperature of 24 ± 1°C throughout the experiment by a room air conditioner. Aquaria were aerated using an air stone.
supplementation. The test diets were prepared with supplementation of 17α-MT (50 and 100 mg · kg -1 ) and E 2 (50 and 100 mg · kg -1 ) following the methods of Degani (1985) and Santandreu and Diaz (1994). The feed was then stored in tightly sealed plastic bags and placed in a freezer at -20°C. The control diet was sprayed with a steroid-free 95% ethyl alcohol solution, processed in the same way as the treated feed, and stored until the day of use. During the entire trial period, a 12 h light-12 h dark photoperiod was maintained. Water temperature was measured daily and recorded as 26 ± 1°C, while pH, dissolved oxygen, and NH 4 + values were evaluated weekly and maintained at 6.5-8.5, > 5 mg · L -1 and 2-5 mg · L -1 , respectively.
Color measurements. The color measurement of fish skin (L * , a * , b * values) (Hunt 1977;CIE 1986;Nickell and Bromage 1998;Berns 2000) from around the dorsal section was performed using a colorimeter (Konica Minolta CR 400) at the beginning (0 th day), 30 th day, and the end (60 th day) of the experiment. The values of the earlier mentioned variables can be explained as follows: L * = lightness (also referred to as luminance); the lightness or darkness of a color. It ranges from 0 (pure black) to 100 (diffuse white), a * = red to green; (+) positive: redness, (-) negative: greenness, b * = yellow to blue (+) positive: yellowness, (-) negative: blueness. Chroma (C * ) (calculated with a * and b * values) indicates the intensity and lightness (clarity) of the colors.
The hue (H°a b ), also calculated with a * and b * values, is an angle value representing the shade of redness, yellowness, greenness, and blueness of the skin color. Hue degree indicates red as it approaches 0°, yellow as it approaches 90°, green as it approaches 180°, and blue as it approaches 270°.
ΔE is the color difference value and was calculated with L * , a * , and b * measurements obtained at the beginning and the end of the trial (Hunt 1977;CIE 1986;Nickell and Bromage 1998;Berns 2000).
Where L t is lightness at the end of the experiment, L 0 is lightness in the initial of the experiment; a t is red to green degree at the end of the experiment, a 0 is red to green degree in the initial of the experiment; b t is yellow to blue degree at the end of the experiment, b 0 is yellow to blue degree in the initial of the experiment.
Growth parameters. Growth parameters of experimental fish were calculated with the following equations: The specific growth rate (SGR) [%· day −1 ], the weight gain (W G ) [%], the survival rate (S) [%], the Feed Conver-sion Ratio (FCR), and Fulton's condition factor (K) were calculated using the respective formulas: where W 0 and W F are the initial and final body weight and t is the time (feeding period) in days; N t is the number of fish at the end of experiment and N 0 is the number of fish at the beginning of the experiment); W F is the weight [g] of the food given and L T is the total length [cm] of the fish.
Sex parameters. In the experiment, the sex reversal of the fish was determined by looking at the primary and secondary sex characteristics.
Statistical analyses. Statistical analyses were conducted with "Minitab Release 17 for Windows" software. Data were subjected to one-way analyses of variance (ANOVA), followed by the Tukey test at a significance level of 5% (0.05).
Ethical note. This research was approved by the Ethics Committee of Sinop University with Reference No. MYO-1901-16-22. All the procedures applied in this study took into account the importance of preventing or at least minimizing, any kind of animal discomfort or suffering.

Results
At the end of the 60-day feeding period, it was observed that the fish group fed a higher dose of 17α-MT (100 mg · kg -1 ) had a significantly positive effect (P < 0.05) in terms of weight increase, weight gain, FCR, and SGR. Growth parameters in the group fed 17α-MT (50 mg · kg -1 ) were also better compared to the group without 17α-MT. However, no change was observed in the fish groups fed E 2 . The highest survival rate was observed in the control group (93.33 ± 2.45%) followed by 50 mg · kg -1 17α-MT treated group (86.67 ± 7.35%) (P < 0.05). The survival rates of the remaining fish groups (50-100 mg · kg -1 E 2 and 100 mg · kg -1 17α-MT) were low.
In terms of sex reversal, the fish in 17α-MT groups exhibited 100% masculinization, whereas in E 2 supplemented fish groups (50 and 100 mg · kg -1 ), the feminization rates were 88.88% and 93.33%, respectively (Table 2). No intersex fish were found at the end of the experiment.
Color analysis (instrumental) in terms of L * , a * , and b * values showed that the group fed 17α-MT displayed brighter coloration compared to other groups (P < 0.05). In terms of Hue (H°a b ) values, the skin color of fish in 17α-MT supplemented groups showed more intense shades of red, while the skin colors of fish in E 2 supplemented groups were observed to be shades of grey-brown closer to the shade of yellow color. The maximal change in color difference (ΔE) was observed in fish groups supplemented with 50-100 mg · kg -1 17α-MT (Table 3).

Discussion
In the ornamental fish industry, it is economically advantageous to produce certain species that have high commercial value and more demand depending on size, color, and sex. Therefore, in this study, the most commonly used steroids (namely, 17α-MT, and E 2 ) were supplemented to the feed of emperor red cichlid.
Fish groups fed 17α-MT hormone had a better growth rate. These results are in agreement with the studies on In the presently reported study, fish groups fed E 2 had no significant changes in growth parameters, which is in agreement with the results studied on Amatitlania nigrofasciata (see George and Pandian 1996) and Centropomus undecimalis (see Carvalho et al. 2014). Similarly, studies on Xiphophorus helleri by Lim et al. (1992) and Tamaru et al. (2009) stated that the hormones did not have any adverse effect on the growth.
Apart from beneficial effects, the most pronounced negative effect of hormone supplementation is on the sur- Values (mean ± standard error of data for triplicate groups) with different superscripts in the same row are significantly different (one-way ANOVA and Tukey multiple-range test, P < 0.05). SGR = specific growth rate, FCR = feed conversion ratio, K = condition factor. Values (mean ± standard error of data for triplicate groups) with different superscripts in the same row are significantly different (one-way ANOVA and Tukey multiple-range test, P < 0.05); L * = Lightness (also referred to as luminance); the lightness or darkness of a color; a * = red to green; (+) positive: redness, (-) negative: greenness, b * = yellow to blue (+) positive: yellowness, (-) negative: blueness; ΔE is the color difference value and was calculated with L * , a * , and b * measurements obtained at the beginning and the end of the trial; Chroma (C * ) (calculated with a * and b * values) indicates the intensity and lightness (clarity) of the colors; Hue (H°a b ), also calculated with a * and b * values, is an angle value representing the shade of redness, yellowness, greenness, and blueness of the skin color.
vival rate, which, in turn, is dose-dependent, as well as species and sex specific. In this study, the survival rate was found to be high in the control group and the fish group supplemented with the low dose (50 mg · kg -1 ) of 17α-MT. On the other hand, the survival rate was low in all fish groups supplemented with E 2 and the fish group treated with a high 17α-MT ratio. It has been reported that the high concentration of 17α-MT and E 2 hormones lower the survival rate of swordtail (Tamaru et al. 2009) and zebra cichlid (George and Pandian 1996). Moreover, in the study of Komen et al. (1989) on Cyprinus carpio, the survival rate of fish treated with 25, 75, 125 mg · kg -1 E 2 hormone varied between 51 and 69%, while the fish groups treated with 50 and 100 mg · kg -1 17α-MT hormone had survival rates of 28% and 39%, respectively. In several studies involving the use of 17α-MT, the survival rate was negatively correlated in a dose-dependent manner as evident in Trichogaster lalius (see Katare et al. 2018), O. niloticus (see Das et al. 2010), and Poecilia sphenops (see George and Pandian 1998). Basavaraju et al. (2008) supplemented C. carpio fry and adults with 17α-MT and determined that compared to the control, the survival rate of the fry was lower, while that of the adults was higher.
When the sex ratios were examined, it was determined that the masculinization rate was 100% in fish groups with 17α-MT hormone. The groups supplemented with E 2 had a positive impact on feminization compared to the control group, although it was not as effective as the 17α-MT in masculinization. George and Pandian (1996) supplemented the zebra cichlid with 17α-MT and E 2 and observed the highest male (55%) and female (100%) rates in groups fed 200-300 mg · kg -1 hormone additives. Katare et al. (2018) determined that the 17α-MT supplementation at 50-100 mg kg -1 was insufficient for the masculinization of T. lalius. Furthermore, in a study carried out on swordtail, it was reported that the female rate in the control group was 74.5% and the optimum dosage for feminization (97%) was 400 mg · kg -1 Es (Tamaru et al. 2009). The optimum 17α-MT supplementation rate for masculinization was reported to be 60 mg · kg -1 in O. niloticus (see Das et al. 2010 On the contrary, 17α-MT supplementation did not cause any significant difference in rosy barb and dwarf gourami (Ramee et al. 2020), as well as in C. carpio (see Basavaraju et al. 2008). The findings of the presently reported study suggest that hormone supplementation positively impacts the production of all male fish, thereby fetching a higher market value and increased profitability. This will result in further improvement of the ornamental fish market with the help of mono-sex fish production.
The visual appeal and color of aquarium fish are important criteria for their marketability. In the presently reported study, enhancement in coloration of fish caused by hormone treatment was statistically demonstrated with the applied method and supported by the results of analyses. According to color analyses conducted at the beginning of the study, hue (H°a b ) values indicated shades of grey-brown as the fish were at the early developmental stage. The color differences amongst groups were apparent according to the analyses carried out on day 30. At the end of the study, the desired coloration was determined in 17α-MT-supplemented fish groups and there were differences between these groups over control. However, no such significant improvement in coloration was seen in the groups supplemented with E 2 . As male individuals of emperor red cichlid appear as bright red, the primary reason for enhanced coloration in fish groups supplemented with 17α-MT hormone may be attributed to efficient masculinization in these groups, as it caused production of 100% male population. Dananjaya et al. (2020) supplemented Carassius auratus with various doses (0, 50, 100, 200, and 250 mg · kg -1 ) of annatto (Bixa orellana) and determined that, according to L * , a * , and H°a b values, the efficacy of annatto increased in a dose-dependent manner. Ninwichian et al. (2020) administered three different natural sources of carotenoids (Phaffia rhodozyma, Paracoccus sp., Haematococcus pluvialis) to C. carpio and determined that the fish supplemented with natural carotenoid sources displayed a higher red color in terms of a * and H°a b values. Moreover, Gouveia et al. (2003) used various synthetic and natural astaxanthin, Chlorella vulgaris, Arthrospira maxima (Spirulina), and Haematococcus pluvialis coloration supplements in Kawari and Showa fish, varieties of C. carpio and observed significant effects on the shade of red (a * and H°a b ) according to colorimetric analyses. Larsson et al. (2002) administered 17α-MT hormone to P. reticulata and reported that an intense coloration was observed after the 17 th day and the difference was statistically significant. Similarly, it has been reported that the 17α-MT hormone increased pigmentation which has a positive impact on coloration in B. splendens and Xiphophorus hellerii (see Jessy and Warghese 1987). When the physical color analysis results of the Karsli et al. (2018) study on electric yellow cichlid were evaluated, it was determined that the best coloration was obtained in 17α-MT groups, similar to this study. In contrast to these studies, it was reported that 17α-MT supplementation for 28 days did not affect coloration in Xiphophorus hellerii (see Koshio et al. 1993;Yanong et al. 2006).

Conclusion
In the presently reported study, the juveniles of emperor red cichlid (A. nyassae) which were supplemented with 100 mg · kg -1 17α-MT displayed the highest growth and weight gain. On the other hand, group E 2 was found to be effective in sex reversal, similar to group 17α-MT. When the results of growth, coloration, sex reversal, and survival rate pa-rameters are analyzed in this study, they were found to be important in the ease of marketing in the ornamental fish trade. Especially, skin coloration is the leading factor that determines the market value of ornamental fish. The discoloration and faded color in aquarium fish cause adverse effects on marketing. Aquarium fish cannot synthesize color pigments sufficiently in the culture environment and thus supplementation of synthetic or natural color through feed should be provided. When the results of growth, coloration, sex reversal, and particularly survival rate are evaluated, the most effective hormone dose for emperor red cichlid fish was determined to be 17α-MT at 50 mg · kg -1 .

Acknowledgment
The author is grateful to Assist. sDr Dilek Şahin and Assist. Dr Meryem Öz for contributing to this research.

References
Ajiboye OO, Qari R, Yakubu AF, Adams TE (2015)    Recently, a younger specimen of S. doederleini was collected from deep water by hook and line from off Fugang, Taitung, southeastern Taiwan. The finding reveals a southward extension of its distribution from cold or temperate into tropical water. A detailed description of this specimen is provided.

Materials and methods
Methods for counts and proportional measurements followed Hubbs and Lagler (1958). Standard length is expressed as SL. Standardization of morphometric data is expressed as a percentage of SL. All measurements were made with digital calipers to the nearest 0.1 mm. The cheek depth is the narrowest space between the lower margin of the eye and the lower margin of premaxilla. Osteological characters were determined by X-radiographs. The specimen is deposited at Pisces Collection of the National Museum of Marine Biology and Aquarium, Pingtung, Taiwan (NMMB-P).  22°43′N 121°20′E), Taitung, southeastern Taiwan, March 2020, by deep-water longline, ca. 400 m, purchased at local auction.
Body moderately slender, deepest region at about middle of abdomen; less compressed. Dorsal profile smooth, nape slightly raised, dorsal-fin base straight at spinous region, slightly raised at soft-rayed region (Fig. 1A). Head moderately large; dorsal profile slightly more pronounced then ventral; dorsal profile barely convex from tip of snout to dorsal-fin origin ( Fig. 2A). Posterior end of head below base of fourth dorsal-fin spine. Snout moderately long, blunt anteriorly, 1.9 times eye diameter. Eye small. Dorsal surface of skull with some smooth vermicular-like ridges. Middle of anterior portion of nape with long smooth ridge, its anterior portion branched (Fig. 2B).
Mouth moderately large, terminal, its gape slightly oblique, forming ca. 10° angle with horizontal body axis; lower jaw slightly overhanging upper jaw; posterior end of maxilla extending to vertical through middle of eye. First suborbital bone (lacrimal) well developed, slightly overlapped with anterior portion of maxilla, its lower margin smooth. Two nostrils well in front of eye. Anterior nostril rounded, bearing high rim with flap, its broader reaching anterior margin of posterior nostril when reflected. Posterior nostril elliptical, horizontal axis longer and slightly above the anterior nostril, with very low flashy rim along its lower margin ( Fig. 2A).
Opercle with strong spine, pointed and rather distinct, mostly exposed from skin and extending to slightly overhanging posterior margin of opercle (Fig. 2C). Exposed margin of preopercle with strong stout spines, those along angle stronger and longer (Fig. 2D). Posterior margins of upper portion of subopercle smooth; lower margin of interopercle smooth. Posttemporal exposed, with weak serrations on posterior margin; upper portion cleithrum largely exposed; supracleithrum not exposed, covered by operculum totally.
Single dorsal fin with long base, deeply concaved at posterior third, spinous portion strongly arched with its base longer than that of soft-rayed portion, base of spinous portion 2.4 times base of soft-rayed portion (Fig.  3A). Dorsal-fin spines somewhat compressed; first spine short, the fourth spines longest, distinctly longer than third spine, rest spines gradually shorter posteriorly, except for last spine (11 th , also leading spine of posterior lobe), which is slightly longer than penultimate spine. Second dorsal-fin ray longest, gradually shorter posteriorly.
Pectoral fin moderately large, long oval (Fig. 3B), its origin slightly below body axis and lower opercular spine, its tip reaching base of 9 th dorsal-fin spine when addressed. Origin of pelvic fin (Fig. 3C) below origin of pectoral fin, about vertical through base of third dorsal-fin spine; its tip slightly behind middle of interspace between origins of pelvic and anal fins, its tip well before anus when pressed. Anal fin small with short base, first spine very short, all three spines shorter than second ray (also longest) (Fig.  3D); outer margin of anal fin straight. Caudal fin truncate, very slightly emarginated as middle (Fig. 3E).
Teeth villiform on jaws, vomer, and palatines; no teeth at symphysis of upper jaw. Vomer with wide triangular patch of small villiform teeth; no backward prolongation of vomerine tooth patch. Palatine with a narrow band of numerous rows of small teeth. No teeth on pterygoids or tongue. Pseudobranch present.
Gill rakers on first gill arch 3 (rudimental) + 1 (developed) on upper limb, 7 (developed) + 6 (rudimental) on lower lobe. Scales weakly ctenoid, including those on cheek, interorbital, operculum, chest, nape, chest, and base of pectoral fin. No scale on snout and chin. Predorsal scales beginning in plane about halfway between posterior margin of orbit and upper end of gill slit.
Lateral line complete, lateral line scales not enlarged, each with simple sensory tubule. Rows of scales above and below lateral line parallel to it. Bases of soft-rayed portions of dorsal and anal fins covered by small scales. Pectoral and caudal fins with scales basally.
Coloration. When fresh, body yellowish brown dorsally and grayish ventrally with all fin darker, except for membranes of spinous dorsal fin translucent; 5 broad, irregular white stripes on lateral side of body; white bar on head and cheek right behind eye; soft dorsal fin and caudal fin with bright white margin. Body dark brown in general, with 5 broad pale stripes, uppermost one along dorsal-fin base, and other 4 on lateral side of body; fins darker, except for membranes of spinous dorsal fin pale; posterior margins of soft dorsal fin and caudal fin pale.
Remarks. The Taiwanese specimen is identified as Stereolepis doederleini based on the distinct coloration with 5 broad pale stripes on lateral sides of body among the family. It differs from the only other congener, S. gigas, co-occurring in the northwestern Pacific, by having the spinous dorsal fin distinctly higher than the soft dorsal fin (vs. the former distinctly lower than the later in S. gigas); the caudal fin truncate (vs. slightly concave or emarginated); 5 pale stripes on lateral sides of body (vs. black patches on lateral sides of body); lateral-line scales less than 73 (vs. about 80), scale rows below lateral 28-31 (vs. 35-40), and posterior end of maxilla reaching a vertical through posterior margin of eye (vs. reaching a vertical through anterior margin to middle of eye) (Mochizuki 1984; this study).
It is notable that Mochizuki (1984) reported 57-68 pored lateral-line scales for S. doederleini, whereas we counted 73 (including 3 on caudal-fin base) for our specimen which may represent the individual variation.

Discussion
The presently reported material of Stereolepis doederleini was caught by hook-and-line from deep water off Fugang fishing port, Taitung, eastern Taiwan. A portion of the local fisheries there are targeting high economical value demersal species such as Doederleinia berycoides (Hilgendorf, 1879) and Atrobucca nibe (Jordan et Thompson, 1911) and it is likely that S. doederleini was a bycatch with these species by deep water line fishery, where the habitat is assumed to be deep sandy or muddy sea floor (CNT, personal observation).
The discovery of Stereolepis doederleini in eastern Taiwan may represent a rare dispersal case of a cold-water species to southern and warmer regions. Similar cases were documented by Koeda and Muto (2019) and Chou and Tang (2021), where cold water species Pholis fangi (Wang et Wang, 1935) and Sebastes thompsoni (Jordan et Hubbs, 1925) were found in local catches of southwestern and northern Taiwan, respectively. Still, given the lack of further specimens, it remains uncertain whether the presently described specimen represented a rare local population or a rare dispersal event from the high latitude region.

Introduction
The family Ariidae accommodates more than 140 species of catfishes, found mainly in marine and brackish waters (Froese and Pauly 2021). However, some species are euryhaline and can live in a wide range of environ-ments, from freshwater to marine, including the spotted catfish, Arius maculatus (Thunberg, 1792), which is widely distributed across the Indo-West Pacific Region (Rainboth 1996). This species has been reported to grow as large as 80 cm TL, but the typical maximum size is 30 cm TL (Froese and Pauly 2021). The trophic level Acta Ichthyologica et Piscatoria 51 (4) of A. maculatus is estimated to be 3.4 ± 0.46, indicating carnivorous behavior. This fish feeds mainly on benthic invertebrates (Angsupanich et al. 2005), and plays an important role in the ecosystem; an imbalance in its abundance can trigger a trophic cascade (Froese and Pauly 2021). This fish is targeted for harvest in many countries, where the fisheries operations are mostly in either marine or brackish water environments (Arshad et al. 2008;Chu et al. 2012;Jumawan et al. 2020;Kutsyn et al. 2021). There is also a unique inland fishery for this species in Songkhla Lake, the largest natural lake in Thailand. The catch of A. maculatus was over 180 metric tons in 2011, following the restoration of the lake's fishery resources by declaring fish sanctuary zones and a stocking program for some indigenous species (Tanasomwang and Assava-aree 2013). The recent catch of this species in 2020 increased tremendously to 330 metric tons (Pattalung Inland Fisheries Research and Development Center, unpublished data), which was almost 1.5-fold higher than the previous record. This sharp rise in harvest implies that appropriate fisheries management of this resource is urgently required to prevent a collapse of this spotted catfish stock.
Effective fisheries management requires an understanding of the stock status and population dynamics of the targeted fish species, where the growth parameters (i.e., asymptotic length and curvature parameter) are among the crucial inputs (Isley and Grabowski 2007; Katsanevakis and Maravelias 2008). Accurate estimates of growth parameters are important for monitoring the stock status as well as for assessing management actions that have been applied to maintain the integrity of the fish stock (Zhang et al. 2020). To estimate growth, precise and accurate age data are required. Age can be estimated by several methods but counting natural growth rings of hard body parts is the most common and is generally reliable (Vitale et al. 2019). Many calcified structures, both inner parts (e.g., otoliths, vertebrae, and urohyal bone) as well as outer parts (e.g., scales, opercular bones, spines, and fin rays) have been shown to reliably reflect the age of fishes (Campana 2001;Morioka et al. 2019;Phomikong et al. 2019). As many hard parts are available for age determination, a comparison of multiple structures to determine the most suitable ones for a particular species or population is always recommended (Zhu et al. 2017). Khan et al. (2011) mentioned that selecting the most suitable hard part for aging is one of the problems in age and growth studies of fishes. They suggested comparing different bony structures of the targeted fish to obtain the most suitable one that has high precision and low aging error before further growth analysis. Moreover, if there is no significant difference in age reading between the inner and outer parts, the use of outer parts is recommended, since the fish does not need to be sacrificed; this is particularly desirable for long-lived species (Zhu et al. 2017).
Along with selecting suitable hard parts for aging, the most suitable model must be chosen for the length-at-age key, which can be determined by the shape of the growth curve and error component of the data (Zhu et al. 2017). Numerous growth curves, as well as their representative models, have been introduced and applied to explain fish growth. Growth models relate the age of fish in a population to their length or weight; the von Bertalanffy growth model is the most commonly adopted and widely used (Jones 2000;Katsanevakis and Maravelias 2008;Zhu et al. 2017). The von Bertalanffy model is commonly used because of the shape of the desired growth curve and from biological assumptions, one of which is that fish growth slows with age (Katsanevakis 2006). Other commonly used models include the Gompertz and logistic growth models (Katsanevakis 2006;Zhang et al. 2020). Both models are well suited to fishes, which exhibit low initial growth rates, but the regions above and below the inflection are asymmetrical in the Gompertz model, and symmetrical in the logistic models (Quist et al. 2012). The base assumption of all three of these models is that fish show asymptotic growth (Katsanevakis and Maravelias 2008).
The aim of this study was to provide length-at-age data and a growth model for the A. maculatus stock in Songkhla Lake, Thailand. We assess the suitability of hard parts for aging and evaluate three growth models to describe the relation between age and length, which can be used as a reference for other A. maculatus stocks. The results are also expected to be further used for stock assessment and fisheries management of the stock in Songkhla Lake for its sustainable exploitation.

Materials and methods
Study area and fish sampling. Songkhla Lake (Fig. 1) is a shallow coastal lagoon in southern Thailand (07°24′08″N, 100°15′42″E), with the mean depth of 2 m. The water body covers 1018 km 2 and is classified into three distinct zones: upper (459 km 2 ), middle (377 km 2 ), and lower (182 km 2 ). This lake hosts a high diversity of fishes (ca. 450 species), due in part to its range of physiological characteristics. For example, the upper zone is a freshwater environment, while the lower zone is brackish (maximum 15‰) at the mouth, where it enters the Gulf of Thailand (Damchoo et al. 2021). Meanwhile, the salinity in the middle zone fluctuates between 10‰ and 15‰ as it is a mixing zone between fresh and saline water (Tanasomwang and Assava-aree 2013). Samples of A. maculatus were collected from four landing sites (Fig. 1) in the upper and middle zones of Songkhla Lake (where the fishery is intensive) between January and December 2020. Individual fish were labeled, measured for standard length (SL), fork length (FL) total length (TL) to the nearest 0.1 cm, and weighed to the nearest 0.01 g in situ. Samples were then packed in ice and taken to the Pattalung Inland Fisheries Research and Development Center. Additionally, 15 A. maculatus representing various size classes were collected every three months for annual ring validation.
At the fisheries center, the largest pair of otoliths (i.e., lapilli) were removed, washed, and kept dry in a vial. Dorsal-and pectoral-fin spines were cut with bone-cutting forceps. Each hard part was embedded in resin and cut by a low-speed diamond saw (South Bay Technology Inc., model: 650). Bony parts were then polished by sandpaper (grit size ranging from 600 to 1500) until the core was seen. Each polished sample was photographed under 40× magnification, and annual rings were counted visually from the monitor and using the Image-J program. An annual ring was considered as the boundary between the inner edge of a wide opaque zone (i.e., corresponding to high growth rate) and the outer edge of a narrow translucent zone (i.e., corresponding to low growth rate) (Kutsyn et al. 2021). Three (3) readers, with no background information of the size of each individual fish, were assigned for aging each sample.
Data analysis. Length-length relations and length (TL)-weight relation (LWR) were examined by linear and curvilinear regressions. The estimated parameter "b" from LWR was tested for significant deviation from 3 by using a t-test. where R is the radius (distance between the center and the edge of hard part), R n is the distance from center to outer edge of last complete band, and R n -1 is the distance from center to outer edge of next-to-last complete band. The difference in MIR among months of sampling was tested by Kruskal-Wallis test, and Dunn's post test was applied when a significant difference was found at α = 0.05. The percentage of agreement (PA) among the three readers was calculated as the ratio of the number of agreements among the three readings to the total number of readings made. Precision in age reading among the three readers of each hard part was tested by two methods (Campana 2001): mean percentage error (MPE) 1 and coefficient of variation (CV).
where MPE j is the mean percentage error for the j th fish, x ij is the i th age estimate of the j th fish, x ̅ j is the mean age estimate for the j th fish, R is the number of times that each fish was aged, and n is the number of samples. where CV j is the coefficient of variation for the j th fish. The age readings of each fish sample and each hard part from the three readers were then averaged and rounded to the nearest integer. Two additional readers with extensive experience in fish aging reviewed each hard part and checked its designated age. The age-bias plot (Campana et al. 1995) was applied to visually assess potential aging differences among hard parts. Finally, three common growth models (Duarte-Neto et al. 2012), i.e., von Bertalanffy model (von Bertalanffy 1938), Gompertz model (Gompertz 1825), and Logistic model (Ricker 1975) were fitted to the observed length-at-age data: where L t is the predicted length-at-age t, L ∞ is the asymptotic length, k is a relative growth rate parameter, and t 0 is the age when length is theoretically zero. The growth models were fitted to length-at-age data using nonlinear least-squares. The residual sum of squares (RSS) was used to measure the discrepancy between the data and an estimation model. The growth performance index (Ø′, Pauly and Munro 1984) of each growth model was estimated by The obtained L ∞ [cm] and k values were further used to estimate the natural mortality coefficient (M) by using Pauly's equation (Pauly 1980) log 10 M = −0.0066 − 0.279log 10 L ∞ + 0.6543log 10 k + 0.4635log 10 T where T is the annual mean water temperature, which was set at 30°C (International Lake Environment Committee Foundation 2021). Data analysis was conducted by using R-statistics (R Core Team 2020) under Package FSA (Ogle et al. 2021) and "fishmethods" (Nelson 2021).

Results
A size distribution of the 213 A. maculatus samples used in this study is presented in Fig. 2, with a range between 35 and 238 mm TL (mean ± SD 128 ± 43 mm TL). Body weights ranged between 0.4 and 154.1 g, with mean ± SD of 26.4 ± 23.7 g. The length-length relations showed high correlation between measurements (R 2 > 0.95; Fig. 3A-B); meanwhile, the log-transformed LWR revealed positive allometric growth, i.e., parameter "b" of WLR was significantly higher than 3.0 (P < 0.01; Fig. 3C).
The 60 A. maculatus samples for the MIR study ranged between 35 and 238 mm TL, with the mean ± SD of 128 ± 43 mm TL. The MIR results showed clear increasing trends from January (MIR near 0.5) to October (MIR near 1.0) in all three hard parts, and a significant difference was found between January and the other sampling months (P < 0.05; Fig. 4). The highest MIR was found in October for all hard   parts and was almost double the MIR in January. The largest difference in MIR values between these two months was observed in otoliths. The significantly lower MIR in January implies that an annulus is deposited once a year.
Age estimates from the three hard parts ranged from less than 1 to a maximum of 6 years (Fig. 5). The percentage of agreement among the three readers was highest for otoliths (58.2%), resulting in the lowest MPE (9.5%) and CV (12.6%). Meanwhile, the pectoral-fin spine samples showed a higher percentage of agreement (and hence lower MPE and CV) than dorsal-fin spine samples (Table 1).
The designated age of each hard part (i.e., by three readers in the first round) was checked by two highly experienced readers with a perfect agreement and thus was considered the observed age of each individual fish ( Table 2). Biases of age reading from otoliths and spines were obvious at ages beyond 3 years, as the observed age from otoliths was a bit higher than the observed age from spines; meanwhile, less bias was found between the two spines (Fig. 6).
Parameter estimation in all models using the observed length-at-age data from the three hard parts is displayed in Table 3. It is clear that the asymptotic length (L ∞ ) estimated from the von Bertalanffy model (around 290 mm TL) was higher than from the other two models, for all hard parts. The larger L ∞ from the von Bertalanffy model was compensated by low k values, which consequently made Ø′ values for the three models relatively similar. The estimated natural mortality coefficient fluctuated between 0.573 and 1.631 year -1 , due to the estimated L ∞ and k values. In each growth model, the lowest sum of squares was obtained from the length-atage data from otoliths. Therefore, this hard part was used to explain the growth of A. maculatus in Songkhla Lake as    L ∞ = the asymptotic total length, k = relative growth rate parameter, t 0 = age when length is theoretically zero, RSS = residual sum of squares, Ø′ = phi prime value, M = natural mortality coefficient. By applying the growth curves of the three models (Fig. 7), the estimated lengths at age 1 to 5 years were almost identical, whereas the estimated sizes from the von Bertalanffy model were larger at age 6 years old and above relative to the other models.

Discussion
The age-length key and growth estimation of fishes and shellfishes can provide valuable insight into their life history and be further used to prescribe optimum fishing regulations for sustaining their fisheries (Isley and Grabowski 2007). In this study, the age-length key and growth of A. maculatus were assessed for the population in Songkhla Lake, Thailand. All the samples were from the upper or middle zones of the lake, implying that they can develop a stock in a low-salinity or freshwater habitat, contrary to the suggestion by Kutsyn et al. (2021) that this fish avoids the freshwater environment in the Mekong Delta.
The maximum length of A. maculatus in this study was 23.8 cm TL, which is similar to the maximum size from Age validation through MIR analysis provides relative certainty of annulus formation and confirms the growth zone deposition in A. maculatus. Low MIR was found in the early part of the year (January, winter) when the water temperature in the inner zone is normally less than 27°C; it is around 30°C during the rest of the year (Anonymous 2021). The maximum age (6 years) of A. maculatus found in this study is within the applicable range for age validation. MIR is not suitable for long-lived species  Growth curves of Arius maculatus in Songkhla Lake, Thailand, from three growth models. [year] or older individuals (i.e., more than 10 years) (Campana 2001). A higher percentage of agreement in aging from otoliths than with other hard parts has also been reported by other researchers (Khan et al. 2011;Zhu et al. 2017). The percentage of agreement was 50%-60% in this study. Other studies that employed only two readers achieved rates of over 60% (e.g., Khan et al. 2011;Gebremedhin et al. 2021). Although the percentage of agreement was lower in this study, verification among the three readers through MPE and CV was still lower than the upper limit of 20% (Winter and Cliff 1996;Cruz-Martínez et al. 2004). More age bias was observed between otoliths and spines (both types) than between dorsal-and pectoral-fin spines. In older fish (i.e., beyond 3 years), the higher ages estimated from otoliths could be because the spine nucleus of many fish species is reabsorbed and replaced by a hole (i.e., vascularization), which eliminates or obscures the first annulus, causing underestimation of age from spines (Drew et al. 2006;Khan et al. 2011). For this reason, together with the greater consistency in aging among the three readers, otoliths were selected for growth estimation, similar to several other studies (e.g., Khan et al. 2011;Zhu et al. 2017;Almamari et al. 2021;Gebremedhin et al. 2021) that compare ages from various hard parts.
The growth models based on the age-length key from otolith reading revealed a lower residual sum of squares than for spines, which confirms the suitability of this hard part for A. maculatus aging. Higher L ∞ and lower k values from the von Bertalanffy model than from other growth models have been reported in many studies (e.g., Cruz-Martínez et al. 2004;Zhu et al. 2009;Duarte-Neto et al. 2012;Zhang et al. 2020). The obtained Ø′ values from all three growth models were within the range of values from stocks of A. maculatus elsewhere (2.08 < Ø′ < 3.14), which were estimated either by otoliths or length-frequency data (Kutsyn et al. 2021). A suitable growth model should not only be selected by mathematical measures (Katsanevakis 2006;Katsanevakis and Maravelias 2008), but also biological as well as management points of view (Zhu et al. 2009;Haddon 2011). In a mathematical sense, the von Bertalanffy model was generally considered the most reliable candidate model for fish stocks (e.g., Zhu et al. 2009;Duarte-Neto et al. 2012;Zhang et al. 2020). From a management viewpoint, lower L ∞ and the higher k values of A. maculatus in Songkhla Lake from the Gompertz and Logistic models would result in an overestimated natural mortality coefficient, which would have drastic implications for fisheries management (Khan et al. 2011). For example, it could lead managers to increase fishing effort due to the high estimated natural mortality.

Conclusion
The age-length relation and growth of A. maculatus in Songkhla Lake, Thailand, were examined by using hard parts. Different aging structures and growth models were compared for biases in the results. Results showed that otoliths provided more precise age estimation among three readers. The von Bertalanffy model was judged to be the most suitable candidate for growth estimation because of the lowest residual sum of squares. The obtained growth parameters (i.e., L ∞ and k) can be used as inputs in other fish stock assessment models (e.g., the yield per recruit model) to investigate the optimum fishing level for this stock, which currently experiences high fishing pressure.

Ricker WE (1975). Computation and interpretation of biological statistics of fish populations. Bulletin of the Fisheries Research Board of
Canada 191: 1-382.

Introduction
The devil firefish, Pterois miles (Bennett, 1828), commonly known as lionfish, is a fish species belonging to the family Scorpaenidae, native to the Indo-Pacific Ocean where it is distributed from the Red Sea to Sumatra. It is also present in the Mediterranean Sea which it most likely entered from the Red Sea through the Suez Canal ). The first record of this species in the Mediterranean dates back to 1991 when a single specimen was recorded from the Levantine coast (Golani and Sonin 1992). Subsequent records appeared after more than two decades when two specimens were collected in 2012 in Lebanon (Bariche et al. 2013). The first signs of an increase in the initially slow-paced expansion were documented in Cyprus when numerous specimens were recorded in the 2013-2015 period (Kletou et al. 2016). Substantial expansion both in abundance and space followed and P. miles spread to many coastal areas including Turkey and Greece in the north and Tunisia and Italy in the west (Turan and Öztürk 2015; Dailianis et al. 2016;Azzurro et al. 2017;Dimitriadis et al. 2020). Until 2019, the northernmost record of this species was indicated by Dimitriadis et al. (2020) near the Greek island of Corfu in the Ionian Sea. Subsequently, sightings of the lionfish were recorded in the Adriatic Sea near Lecce (Italy) and beach Dhermi (Albania) in July 2019 and in Torre Canne near Brindisi in August 2020 (Di Martino and Stancanelli 2021). Dynamics of the expansion in the eastern Mediterranean and invasive potential of P. miles led Karachle et al. (2017) to foresee its potential arrival in Albanian waters a few years prior to its first record in the area. The concerns associated with the occurrence of this species in the Mediterranean are mostly motivated by the western Atlantic scenario whereby a non-native lionfish (Pterois miles/Pterois volitans complex) severely impacted the biodiversity and ecological processes in invaded areas. For example, high predation rates of this generalist piscivore species negatively impact local fish communities by reducing their abundance and recruitment at invaded locations (Ballew et al. 2016;Goodbody-Gringley et al. 2019).
In this paper, we report the first observation of this species in the middle eastern Adriatic Sea, in Croatian waters.

Material and methods
A specimen of lionfish was observed and photographed (Fig. 1) by a professional underwater photographer during scuba diving on 13 August 2021 at a depth of 15 m, near Komiža at cape Stupišće (island Vis, Croatian waters, middle Adriatic; 43°0′18.25″N, 016°4′1.27″E) (Fig. 2). Diving was organized by the local diving center "Manta". Sea temperature at the site was measured at 24°C with Mares Genius diving computer.

Results and discussion
The length of the specimen was approx. 13-15 cm in total length. It was observed on the bottom consisting of large boulders overgrown with various algae including invasive Caulerpa cylindracea (Chlorophyta). The individual was slowly moving on the algal mat which allowed the photographer to take high-quality photos. Divers observed the fish at the same position on three consecutive days. Due to the nature of the record (photos and video), it was not possible to accurately count meristic characters which distinguish P. miles and P. volitans, so the species identification was tentatively assigned to Pterois cf. miles. Tentative identification was based on particular morphological traits of the specimen visible in provided photos such as distinctive coloration i.e., alternating brown and white vertical stripes throughout the body; large feather-like pectoral fins; conspicuously large dorsal fin spines and fleshy tentacles above the eyes and mouth; soft rays of dorsal, anal and caudal fins with dark spots (Golani and Sonin 1992;Bariche et al. 2013). If we consider the route of species spreading in the eastern Mediterranean, confidence in the species assignment is further strengthened as many of the records along the route were genetically confirmed as P. miles records Stern et al. 2018;Vavasis et al. 2020).
The area where the specimen was found is located on a protruding cape on a northwestern part of Vis island oriented toward the open sea and influenced by open sea currents (Fig. 2). The location itself is about 143 Nm farther than the closest previous Adriatic record reported from Italy (Torre Canne) by Di Martino and Stancanelli (2021). The present record also represents the northernmost record of this Lessepsian migrant both in the Adriatic Sea and the entire Mediterranean to date. Predictions of the expansion of P. miles in the Mediterranean from various studies offer different future scenarios for the Adriatic Sea (D'Amen and Azzurro 2020; Dimitriadis et al. 2020;Poursanidis et al. 2020). Such differences mostly stem from diverse methodologies and parameters considered in driving the prediction models, but also due to differing time frames considered in predictions. Only the northernmost part of the Adriatic is consistently excluded as a distributional area in these predictions, while predictions for the middle and southern Adriatic vary. For example, the study by Dimitriadis et al. (2020) indicated a scenario with an increased risk of expansion towards the middle Adriatic until 2050. These authors considered the role of low winter temperatures as a limiting factor for the expansion and establishment of the species. On the other hand, for example, Poursanidis et al. (2020) considered the Adriatic to be an unfavorable area for the lionfish invasion based on habitat suitability modeling. Kimball et al. (2004) experimentally determined that at a mean temperature of 16.1°C lionfish stop feeding while a mean 10°C was determined to be a chronic lethal temperature. At present, low winter temperatures in the middle Adriatic (Russo et al. 2012), which fall below the one at which lionfish cease to feed, can pose an obstacle for its successful invasion. However, it is suggested that lionfish can survive prolonged fasting which, in synergy with the possibility of climatic niche expansion and sea warming trends, can challenge this hypothesis (Parravicini et al. 2015;Côté and Smith 2018;Grbec et al. 2018). In any case, the present record clearly shows that the species reached the middle Adriatic, but its establishment and impact will need to be evaluated with future investigations.
As indicated by Azzurro et al. (2017) the majority of records of this species in the Mediterranean were made based on underwater footage provided by divers as is the case with the present record. Recently, Phillips and Kotrschal (2021) took advantage of this fact and, using diving centers' expertise, managed to detect additional lionfish sightings throughout the Mediterranean coast indicating the high potential of such methodology in the tracking of its expansion. Also, citizen science campaigns have been initiated throughout the Mediterranean to keep track of lionfish expansion (Azzurro et al. 2017;Giovos et al. 2019).
Due to the high negative impact of P. miles on native fish communities in invaded areas, campaigns aimed at population control of the species have been initiated in the eastern Mediterranean, particularly in Cyprus where such actions resulted in a significant decrease in lionfish numbers in areas targeted (Kleitou et al. 2021). Actions aimed at population control rather than eradication are more realistic in cases of marine invasions as instances of successful eradication are rare (Giakoumi et al. 2019). As early detection and rapid response in the first stages of invasion are considered crucial for successful mitigation and even eradication (see Campbell et al. 2018), we emphasize the need for immediate action in Croatia which should be coordinated among scientific institutions and relevant governmental bodies. For example, actions aimed at population control should be foreseen and an appropriate legal and logistic basis for such actions should be established as soon as possible. Several citizen science campaigns have already been undertaken in Croatia in the last decade to heighten the general public's awareness about marine biological invasions (for example, in the course of LEKFishResCRO project founded by the Croatian Science Foundation) and to motivate citizens to indulge in monitoring efforts through reporting observations of unusual species. Also, the perils of the lionfish invasion, both in terms of its ecosystem impact and potential health hazard due to its venomousness, have already been conveyed to the general public through various news channels and social networks even before this record and similar efforts should be continued.
An illustration using Mediterranean fish invaders. Ecology Letters

Introduction
Large rivers basins harbor a significant share of the world's aquatic biodiversity, providing important goods and services to the society, including fisheries. Ganga basin is the largest and most important river basin in India. The wetlands of the Ganga basin are mostly formed as a result of the meandering of the river or sloughs or tectonic depressions receiving huge surface runoff or freshwater from the river. The connection of the wetland to the river promotes aquatic biodiversity, especially a higher abundance of small indigenous fishes (Manna et al. 2018). However, the intrusion of exotic fishes into associated wetland exerts invariable threats upon native riverine fish biodiversity (Singh and Lakra 2006). The Ganga basin, which supports a wealthy fish diversity of more than 266 fish species (Talwar and Jhingran 1991), has already been invaded by more than 10 species of exotic fishes in almost its entire freshwater stretch (Sarkar et al. 2012) resulting in considerable damage to riverine fish diversity. The entry of exotics into the riverine system has been presumed mainly to have come about due to illegal or unwanted introduction from aquaculture practices or by other anthropogenic activities, including extreme climatic events like floods, etc. (Raj et al. 2021). Besides exotic carps, which have been legally brought for aquaculture enhancement in India, the Ganga River has experienced in recent years the appearance of a few exotic aquarium species like Gambusia affinis (Baird et Girard, 1853), Pterygoplichthys pardalis (Castelnau, 1855), and Pterygoplichthys disjunctivus (Weber, 1991) (see Singh et al. 2013;Das et al. 2020). However, the occurrence of exotic alligator gar, Atractosteus spatula (Lacepède, 1803) has not been documented from the main channel or any of the water bodies associated with the largest river of the country. Atractosteus spatula, representing the family: Lepisosteidae, is a native to North America primarily from the Mississippi River basin (Raz-Guzmán et al. 2018). Alligator gar has been established worldwide as an outcome of the ornamental fish trade (Salnikov 2010). This highly predatory species has been reported recently from several water bodies of different Indian states like Assam (Anonymous 2020), West Bengal (Thakur 2016), Odisha (Anonymous 2017a), Andhra Pradesh (Vadlamudi 2021), Kerala (Kumar et al. 2019), and Maharashtra (Ghai 2018, Patil et al. 2019) displaying its probable congenial interaction with the varied environmental habitats (Fig. 1). The present paper documents the first record of the alligator gar from an open wetland connected to the Ganga River in West Bengal, India, along with a description of the aquatic environment.

Material and methods
A single specimen of Atractosteus spatula was captured from Chharaganga Beel (wetland) (23°27′48.87′′N, 88°20′46.7592′′E), Nabadwip, West Bengal using a gillnet (mesh 10-15 cm) on 23 July 2020 during the early morning hours of fishing activity. The fish was identified in line with the methodology of Bigelow et al. (1963).
The associated hydrological parameters were determined as a part of the assessment of the associated wetland of the Ganga River following standard methods (Baird and Bridgewater 2017).
Ethical statement. During the study, no harm was made to alligator gar which has been described in this paper. After the study, the fish was released in live condition into secured captivity so that it could not reach natural open water.

Results
The fish collected from Chharaganga wetland was identified as Atractosteus spatula, it weighed 2.25 kg, and was characterized by a long and cylindrical body (Figs 2-3). It had an elongated snout and rounded caudal fin. Both the dorsal and anal fins consisted of 7 rays respectively and were positioned close to the tail with the former slightly posterior to the latter. Body scales were hard, non-overlapping, and diamond in shape. Head and snout were devoid of scales. It could be easily distinguished by two rows of sharp series of teeth on the upper jaw. However, the second row of teeth was positioned within the mouth, which was different from the externally visible teeth. The pectoral fin ray count was 14 and the fin was situated in close proximity to the gill opening. The dorsal surface of the body was dark olivaceous while the lower abdominal portion of the body was characterized by whitish shades.

Discussion
Although the information on its habitat usage is scanty, Atractosteus spatula was reported to dwell in both freshwater and marine water habitats (Goodyear 1967). Reports from Indonesia (Hasan et al. 2020) and Texas (Buckmeier 2008) have provided evidence towards its wide tolerance of salinity gradients which has created an additional advantage for its survival, growth, and possible establishment. The presently described site of fish capture is freshwater in nature; however, since it is located at the head of Hooghly estuary, the lowermost stretch of the main channel of the river experiences minor tidal variations. The mean annual temperature [ºC] of the wetland is 27.87 ± 3.16 (Table 1) which is slightly higher than the temperature (24-26ºC) recorded from Vincente Guerrero Reservoir, Mexico (Garcia de Leon et al. 2001). Additional water quality parameters of the wetland, specifically its salinity (0.0167‰-0.079‰), and depth (mean 4.32 m) are at par with the values obtained from the water body of the USA (Boschung and Mayden 2004) indicating a somewhat agreeable environmental condition required for its survival, spawning, and maturation. Having a connection to the wetland, there remains a high chance of its entry into the river. However, the river water possesses a slightly different water quality status (Table 1). Significant differences in parameters like wa- ter turbidity and sediment soil organic carbon might be attributed to the dense growth of aquatic macrophytes, especially floating water hyacinth and different varieties of submerged macrophytes in the wetland (Table 2). Still, there is a high chance of the species establishing in the river as it is basically a riverine species of the Mississippi River system.
The alligator gar has been presumed to have been released by some aquarium hobbyists deliberately into the wetland which is a routine method to get rid of the pet when it attained a size large exciding the capacity of an indoor aquarium. Other than willful release by the hobbyist, extreme climatic events have often been identified for entry of the exotic fishes into Indian inland open waters (Raj et al. 2021) as a route of entry of alien fishes into the natural ecosystem. In the absence of any such climatic event in the studied area in the recent past, its entry through the aquarium trade may be considered. Aquarium trading has now created a miserable approach towards the non-native introduction of fishes (Krishnakumar et al. 2009) with more than 5000 species traded globally, which in turn has linked more than 200 countries of rapidly growing ornamental industry, associated with the second most popular hobby with about 100 million hobbyists in the world (Chan et al. 2020). Alligator gar has been illegally introduced into the Indian fishery system as it is not highlighted in the list of 92 different aquarium fish species considered for import (Kumar et al. 2019). The encroachment and establishment of several other exotic species, which thereby impact the local fishes, are reported from almost the entire riverine system in India (Knight 2010;Sandilyan 2016). As a consequence of ornamental fish trading, vermiculated sailfin catfish, Pterygoplichthys disjunctivus, has been recorded from various river systems of the country like the Ganga or Cauvery (Panikkar et al. 2015;Das et al. 2020). Suresh   Kumar et al. (2020) has recently reported increasing availability of another ornamental three-spot cichlid, Cichlasoma trimaculatum (Günther, 1867), in the Cauvery River. The C. trimaculatum was ranked as posing a "high" risk of being invasive in the river as determined using the Aquatic Species Invasiveness Screening Kit. The alligator gar, Atractosteus spatula, though being recorded regularly beyond its native area, has not yet been recognized as a potential threat as it was not included in the list of 259 non-native freshwater fish species for which risk assessment has been conducted so far using Aquatic Species Invasiveness Screening Kit (Vilizzi et al. 2021). However, considering its possible potential biodiversity risks, an assessment of the species within the United States beyond its native range was attempted (Anonymous 2017b).
West Bengal occupies a major position in the ornamental fish trade with 55% of total production in India. Gupta (2012) has recorded the availability of 139 species of exotic fishes in the ornamental fish market (widely known as Galiff Street) of Kolkata. However, the presence of alligator gar in the market remained unmentioned. The presently reported survey noted that the trading of live alligator gar is performed extensively year-round in the ornamental fish market of the city. Each specimen is marketed at 700.00 Indian Rupee (=8 Euro) at a size range of 15-17 cm. Due to the aggressive nature of their behavior, alligator gar are marketed and maintained indi-vidually. Besides, as a part of the supply chain marketing system, this exotic fish is also transported to various local aquarium traders within the state. Exotic fishes with recreational and commercial purposes have now become potential threats to natural aquatic bodies. Many such fishes have already invaded rivers, reservoirs, wetlands, etc. and established themselves alongside the declined catch of other indigenous fish species. The invasion of Clarias gariepinus (Burchell, 1822), a highly carnivorous catfish in the Ganga River is one such example (Singh et al. 2013). Large-scale anthropogenic modification of the riverine habitat through water obstruction, pollution, etc. has provided a favorable habitat, allowing a competitive advantage for exotic varieties over native ones (Daga et al. 2016). The Ganga River has also observed an increasing number of barricades over the years, especially in the upper reaches of the river. The availability of the adult alligator gar in an open wetland certainly creates chances for ecological imbalance in the wetland as well as in the connected river by displacing the native fish species spectrum. As little is known about its population dynamics and breeding phenology, data regarding its juvenile availability in the local markets requires more attention. A strategy for developing legal ornamental fish trading is urgently needed. A detailed database on habitat preference, breeding habits, and other biological aspects must be developed before each individual fish being targeted is introduced into the ornamental sector.
Studying fish invasions in more than 1000 river basins on a global scale, Leprieur et al. (2008) opined that  more exotic invasions may occur in river basins of developing countries, including India. India is eighth in the world and third in Asia with 788 freshwater fish species where more than one-fourth is either vulnerable or in the endangered category (Kottelat andWhitten 1996, Gopi andMishra 2015). With an increasing number of dams and barrages, exotic invasion can certainly aggravate the situation by causing a decline of precious fish diversity. The best approach to prevent non-native species is to identify the possible entry points rather than eradication methods. The list of fish species presently being imported into India should be critically revised based on their possibility of establishment and invasiveness, or if they reached natural water by chance. Alligator gar is yet to be included in the list of 14 freshwater invasive fish species compiled by Centre for Biodiversity Policy and Law (CEBPOL), National Biodiversity Authority (NBA) as updated in 2018. A priority list should be prepared including those exotic fishes which have the possibility to become invasive in the future. Existing laws like 'The Biological Diversity Act, 2002' also clearly mentioned about not importing any such fish species into India. The National Biodiversity Strategy and Action Plan has many important suggestions for the control and management of such detrimental invasive species (MOEFCC 2014). Strict monitoring and implementation/enforcement of the law are required but this necessitates national/institutional level monitoring. Awareness among local communities about the detrimental effect of identified invasive species can help to reduce their inclusion for species enhancement in aquaculture or as an ornamental fish to be reared as a pet.

Introduction
The labrid fish genus Suezichthys was originally proposed by Smith (1957) as Suezia (a name preoccupied in Crustacea), and subsequently replaced by Suezichthys (see Smith 1958). Suezichthys is a group of small, sexually dimorphic fishes occurring on sandy bottoms and rocky reefs of the Indo-Pacific and the southwestern Atlantic region. Some of the species are found on deep bottoms or mesophotic reefs, e.g., Suezichthy notatus (Kamohara 1958), which have been collected at deeper than 119 m (Shimada 2013 (Whitley, 1941), although not recognized by Russell and Westneat (2013), was regarded as valid by Kuiter (1993). Suezichthys was redefined by Russell (1985) and Russell and Westneat (2013). It superficially resembles the labrid genus Pseudolabrus, but is distinctive in some osteological characters. Suezichthys is also morphologically similar to Halichoeres, but differs by having cheeks scaly (vs. naked); gill membranes forming a free fold across the isthmus (vs. broadly attached); and anal-fin elements III, 10-12 (mostly III, 10 vs. III, 11-13) (Russell 1985;Russell and Westneat 2013).
In Taiwan, only one species, Suezichthys gracilis (Steindachner et Döderlein, 1887) has been documented (e.g., Shen et al. 1993). It is found in coastal marine habitats, often occurring on shallow reefs and adjacent sandy bottoms. Recently, the first author obtained specimens of a rare labrid from local fish markets, which occurred in the catches of deep-water line fisheries off northern and southwestern Taiwan. This species was subsequently recognized as Suezichthys notatus, the second species of Suezichthys recognized in Taiwan. In this work, a detailed description of specimens of S. notatus from Taiwan, comparisons with S. gracilis, and comments on species of Pseudolabrus from Taiwan are given.

Methods and materials
Counts, measurements, and terminology generally follow Russell and Randall (1981) and Russell (1985), except where indicated. The format of the description generally follows Randall and Kotthaus (1977). Definition of the initial phase (IP) and terminal phase (TP) follow Warner and Robertson (1978). Measurements were taken by digital or analog calipers based on the length of the measured items, recorded to the nearest 0.1 mm. Standard length (SL) and head length (HL) were used throughout. Pectoral-fin counts are given as the total number. Caudal-fin length was measured from the posterior edge of the hypural plate to the uppermost tip of the fin. Osteological characters were determined by X-radiographs. For the determination of associations of the hemal (haemal) arch and caudal vertebrae, the X-radiographs were taken by putting the specimen at an angle of ca. 45 degrees between the lateral surface and the X-ray source. Counts were recorded from the left side of the specimens unless otherwise indicated. Specimens of S. notatus were deposited at the Pisces Collection of National Marine Museum of Biology and Aquarium, Taiwan (NMMB-P).

Suezichthys tripunctatus
(Based on NMMB-P34163) Mouth terminal, horizontal, and small, posterior end of maxilla reaching vertical through posterior nostril; lips moderately fleshy; upper lip with 6 longitudinal plicae and lower lip with 1; upper jaw with two pairs of enlarged canines at front, anteriormost pair of canine largest and recurved, second one about half length of first; 15/16 progressively smaller canine teeth laterally in upper jaw, with inner row of 6 (on left)/4 (on right) small canines behind anteriormost teeth; enlarged and antrorse canine at posterior end of upper jaw; lower jaw with 2 pairs of enlarged anterior canines, second canine slightly shorter than first; 11/13 progressively smaller lateral teeth in lower jaw, with inner row of 6/6 pairs of smaller canines behind anteriormost teeth; no teeth on palatine.
Nostrils small, in two pairs just in front of orbit; anterior nostril terminating in small membranous tube; posterior nostril without flap or prominent ridge at margin. Gill membranes not attached to isthmus, forming free fold posteriorly. Gill rakers short, longest less than half length of longest gill filament. Preopercle entire, free posterior margin reaching just above level of lower rim of orbit, free lower membrane extending forward to below level of middle of lower rim of orbit. Opercular membrane broadly rounded, extending posterior to pectoral-fin base. About 26 pores in lateralis system around orbit; ca. 22 pores along free edge of preopercle (based on NMMB-P34163).    Opercle with 6 large scales posteriorly, almost extending to posterior margin of preopercle. Preopercle with 2 rows of cheek scales behind eye, and 2-3 rows below eye, extending forward to beneath middle of lower rim of orbit. Thorax fully scaled, scales about one half to three quarters of size of body scales. Basal portion of caudal fin scaled; forehead, snout, ventral side of head naked; subopercle naked. Body scales large, cycloid.
Dorsal-fin origin above upper pectoral-fin base; analfin origin below first dorsal-fin soft ray; pelvic-fin base slightly anterior to pectoral-fin base. (Fig. 1A-C), body color pink to purple, paler on abdomen. Faint yellow reticulated pattern on lateral body. Group of prominent dark blotches extending from interorbital region dorsoposteriorly. Snout yellow, extending on operculum as two yellow stripes. Three faint yellow stripes gradually narrower extending from behind operculum to caudal peduncle. Dorsal fin pinkish to purple, yellow wavy lines above base and on upper edge of fin. Black spot between membrane of first and second dorsal-fin spine. Anal fin pinkish to purple, its edge yellow. Caudal fin yellow, with   2-3 vertical purple stripes. Pectoral fin translucent pink. Pelvic fin white.

Coloration. (TP individuals) When fresh
When preserved (Fig. 2), body pale. Prominent black blotches or spots on forehead and dorsal fin consistent with fresh condition. Forehead and dorsal-fin base dusky. Few faint dusky lines along, above and below lateral line. Edge of anal fin black.
Distribution. Suezichthys notatus is known from scattered localities across the Pacific Ocean and the easternmost Indian Ocean. Its type locality is Okinoshima, Kochi Prefecture of Japan. Suezichthys notatus also occurs in deep waters off northwestern Australia and Oahu, of the Hawaiian Archipelago. In the Hawaiian Archipelago, S. notatus has been observed along the edges of sand patches near irregular, hard surfaces, and foraging by picking at objects on rocks and sand at depths of 119-272 m (Russell 1985;Chave and Mundy 1994). The Taiwanese specimens described here were caught off the coast of Keelung, near the "Northern Three Islands", at depths of ca. 100-150 m, and off Kaoshiung, southwestern Taiwan (depth unknown); its habitat was assumed to be deep sandy-rocky bottom.
In Taiwan, only two species of Suezichthys are recognized. The Taiwanese specimens of S. notatus and S. gracilis (Fig. 5) differ in various characters: a longer head in S. notatus (2.8-3.0 vs. 3.1-3.4 in SL in S. gracilis); greater body depth at the origin of dorsal fin (3.7-3.9 vs. 4.1-4.7 in SL); greater predorsal length (3.0-3.2 vs. 3.3-3.5); pelvic fin short (2.2-2.5 vs. 1.3-2.2 in HL); pelvic fin of terminal phase individual short, when appressed its tip not reaching the anus (vs. long, with a slightly filamentous extension, when appressed its tip reaching the anus or slightly beyond the origin of anal fin); scale rows above lateral line 2½ (vs. 1½); a low scaly sheath presents on the base of dorsal and anal fin (vs. without).
In coloration, S. notatus and S. gracilis are both overall pinkish and have a black spot present on the fin membrane between the 1 st and 2 nd spine in TP specimens. However, both IP and TP of S. notatus do not possess a lateral stripe on the body (vs. present on both phases of S. gracilis, red in IP and olive-green in TP); presence of black spots on the interorbital region dorsoposteriorly in S. notatus in TP (Fig.  2), or a large black spot at posterior end of dorsal-fin base (vs. none of these present in both phases of S. gracilis).
The morphology of hemal spines of caudal vertebrae differs between S. notatus and S. gracilis from Taiwan (Fig. 4). The hemal arches form a large hemal canal associated with anterior caudal vertebrae of the 10 th to 12-13 th vertebrae in S. notatus (Fig. 4A); in contrast S. gracilis has only a single hemal arch with a large hemal canal, associated with the 10 th vertebra (Fig. 4B). Russell (1985) stated that the hemal arches with large hemal canal are on the 10 th to 12 th vertebrae of S. notatus, but in one of our specimens (NMMB-P34163) the 13 th vertebra also has a large hemal canal.

Abstract
This study assesses the patterns of variation in body shape, and relations of morphological similarity among species of the Theraps-Paraneetroplus clade in order to determine whether body shape may be a trait in phylogenetic relations. A total of 208 specimens belonging to 10 species of the Theraps-Paraneetroplus clade were examined. The left side of each specimen was photographed; in each photograph, 27 fixed landmarks were placed to identify patterns in body shape variation. Images were processed by using geometric morphometrics, followed by a phylogenetic principal component analysis. The phylogenetic signal for body shape was then calculated. To determine the relations in morphological similarity, a dendrogram was created using the unweighted pair group method and arithmetic mean values, while a Procrustes ANOVA and post-hoc test were used to evaluate significant differences between species and habitats. We found three morphological groups that differed in body length and depth, head size, and the position of the mouth and eyes. The body shape analysis recovered the morphotypes of seven species, and statistical differences were demonstrated in eight species. Based on traits associated with cranial morphology, Wajpamheros nourissati (Allgayer, 1989) differed the most among the species examined. No phylogenetic signal was found for body shape; this trait shows independence from ancestral relatedness, indicating that there is little congruence between morphological and genetic interspecific patterns. As evidenced by the consistently convergent morphology of the species in the Theraps-Paraneetroplus clade, the diversification of the group is related to an ecological opportunity for habitat use and the exploitation of food resources. Although no phylogenetic signal was detected for body shape, there appears to be an order associated with cranial morphology-based phylogeny. However, it is important to evaluate the intraspecific morphologic plasticity produced by ecological segregation or partitioning of resources. Therefore, future morphological evolutionary studies should consider cranial structures related to the capture and processing of food.

Introduction
Among Neotropical freshwater fishes, evidence of diversity suggests that allopatric speciation models frequently apply to several clades and that there are few cases of sympatric speciation stemming from adaptive processes (Albert et al. 2020). The main historical processes include river capture and sea-level oscillations, which fragment and merge fluvial networks. In this scenario involving geographic changes and ecological heterogeneity, phenotypic variation has been an important attribute in morphological diversification and environmental adaptation (Albert et al. 2020). Freshwater fish orders such as Siluriformes, Characiformes, Cyprinodontiformes, Gymnotiformes, and Cichliformes are the best examples of morphological diversity in the Neotropical region due to their high species richness and abundance (Albert et al. 2020;Elías et al. 2020).
In Neotropical cichlids, the ability to use new or newly available resources (i.e., ecological opportunity) has been an important mechanism in diversification (Arbour and López-Fernández 2016;Říčan et al. 2016). Studies related to morphological diversity in South American cichlids have demonstrated that body shape and size variation have been the main axes of diversification, most notably the constant presence of morphological convergence between lineages (López-Fernández et al. 2010, 2013. Morphological convergence is interpretable as evidence that natural selection has selected similar traits, thus providing strong evidence for the adaptive quality of said traits (Elmer and Meyer 2011;Losos 2011;Burress 2015).
Despite advances in knowledge regarding the evolution of diverse groups of cichlids worldwide, there are still lineages with incipient research, such as Middle American heroine cichlids. Middle America harbors approximately 124 cichlid species (Říčan et al. 2011;Matamoros et al. 2015) and includes areas considered to be centers of endemism and high diversity, such as the San Juan and Usumacinta ichthyological provinces (sensu Říčan et al. 2016). From evolutionary evidence, it has been assumed that their diversification was promoted by ecological opportunity and resource partitioning (López-Fernández et al. 2012;Burress 2016;Říčan et al. 2016). This is supported by the diversity of body shapes and the specialization of trophic anatomy, particularly of the oral and pharyngeal jaws (Liem 1973;Meyer 1993;Salzburger 2009;Burress 2016). Notably, this has promoted frequent cases of morphological convergence and the low phylogenetic signal of diagnostic characters. This is the main reason for the unclear and complex taxonomy of cichlids in Middle America (Stiassny 1991;Říčan et al. 2008McMahan et al. 2013).
Among Middle American cichlids, the Theraps-Paraneetroplus clade (sensu Říčan et al. 2016) is notable due to the presence of species therein with highly variable and frequently convergent morphology, which is most evident in their body shapes and characteristics associated with food capture (Soria-Barreto and Rodiles-Hernández 2008;Soria-Barreto et al. 2011. This group of fish is estimated to have originated under sympatric conditions approximately 7.3 mya (Miller et al. 2005;Říčan et al. 2016) and is thought to result from the event of ancient adaptive radiation (Arbour and López-Fernández 2016;Albert et al. 2020). The Theraps-Paraneetroplus clade includes 25 species belonging to 10 genera. Moreover, the distribution of this clade is located in the Usumacinta ichthyological province, comprising the hydrological basins of Papaloapan, Coatzacoalcos, Grijalva, Usumacinta, and northern Belize (Říčan et al. 2016).
In addition to ecomorphological evidence indicating that the phenotypic expression of morphological attributes in some clade members is associated with habitat type and feeding (Soria-Barreto et al. 2019), studies on the systematics and evolution of Middle American cichlids have demonstrated the existence of convergent morphological characters between several species of the Theraps-Paraneetroplus clade (López-Fernández et al. 2014;McMahan et al. 2015;Říčan et al. 2016). As the same body shape patterns are recurrent among species that exploit similar habitats Říčan et al. 2016), the existence of lentic and lotic ecomorphological patterns has been previously proposed; species of the genera Cincelichthys, Kihnichthys, Oscura, and Vieja represent the lentic ecomorphotype (i.e., with short and deep bodies), and the species Theraps, Wajpamheros, Chuco, Rheoheros, and Paraneetroplus represent the lotic ecomorphotype (i.e., with elongated and slender bodies). Notably, both ecomorphotypes are present in the genus Maskaheros (see Říčan et al. 2016).
In the Theraps-Paraneetroplus clade, convergent morphological characteristics seem to support the hypothesis of diversification via ecological opportunity and resource partitioning, which contrasts with the hypothesis proposed by phylogenetic systematics and the theory of evolutionary non-independence (Felsenstein 1985). In the non-independence hypothesis, it would be expected that body shapes within the species and genera of this clade would have a diversification pattern similar to that of phylogeny and taxa sharing an ancestor that is most morphologically similar. In this way, species with a lotic body shape should share an ancestor, which should be similarly true for the lentic body shape. In contrast, if the non-independence hypothesis is rejected, then the morphological patterns should not be statistical dependents of the common ancestry (Revell et al. 2008). Therefore, the presently reported study aims to describe and compare the body shape variation patterns in 10 species of the Theraps-Paraneetroplus clade. Furthermore, the phylogenetic signal is obtained to measure the statistical non-independence of the morphologic trait values of the species due to their phylogenetic relatedness (Revell et al. 2008).
For this purpose, geometric morphometrics and comparative phylogenetic methods are used as analytical tools because they are commonly used to study the evolution of biological morphology. Geometric morphometrics can be used to identify variation in the pure shape of organisms, and separate the variation and size of individuals by analyzing shapes in multivariate space (Adams et al. 2004;Zelditch et al. 2004;Aguirre and Jiménez-Prado 2018). Notably, comparative phylogenetic methods can be used to analyze morphological characteristics and their significance in species diversification from a phylogenetic perspective (Pagel and Harvey 1988;Adams and Collyer 2018;Borges et al. 2019;Villalobos-Leiva and Benítez 2020). Recently, phylogenetic approaches have made it possible to understand the significance of morphological variation and changes in the dynamics of biological communities, particularly in speciation, adaptation, and extinction. This is essential to predict the effect of natural and anthropogenic changes on ecosystem processes. It also represents a fundamental step towards the management and conservation of biodiversity on the planet (Cavender- Bares et al. 2009).

Methods
To analyze the morphological variation among members of the Theraps-Paraneetroplus clade, the presently reported study included a total of 208 specimens (females and males of similar size) that correspond to 10 species, representative of each genus in the clade. All the specimens were deposited at the Fish Collection of El Colegio de la Frontera Sur, San Cristóbal (ECOSC). Theraps clade: Chuco intermedium (Günther, 1862) Shape analysis. To reduce morphological variation related to phylogeny and differences in specimen size (allometry), regression of the Procrustes coordinates and centroid size was conducted using the "phyl.resid" function of the Phytools package ver. 0.7-80 in R software (Revell 2009(Revell , 2012. In order to then identify patterns in body shape variation, a phylogenetic principal component analysis (pPCA) was performed based on the Procrustes coordinates of the mean configurations of the 10 species. The nDNA molecular phylogeny based on ddRAD sequences proposed by Říčan et al. (2016) for Middle American cichlids was used in both instances. The body shape variation of the species was displayed on the first three pPCA axes. Additionally, deformation grids were obtained to visualize and describe the morphological variation among species in morphospace. All analyses were conducted in R software using the Phytools package ver. 0.7-80 (Revell 2009(Revell , 2012. Additionally, the phylogenetic signal for body shape was computed by using the Kmult statistic (K) across 1000 permutations via the Geomorph package 4.0.0 in R software (Adams et al. 2021), K values <1 indicate a low phylogenetic signal, while K values >1 indicate a strong phylogenetic signal (Adams 2014). To determine the relation involved with morphological similarity among the 10 species, a dendrogram was constructed using the unweighted pair group method using mean values in Past 4.05 software (Hammer et al. 2001) based on the Mahalanobis distances obtained in MorphoJ 1.07a software (Klingenberg 2011).
Finally, to determine whether significant differences in body shape exist among species and between lentic (LE) and lotic (LO) habitats, a Procrustes ANOVA was used as implemented in Geomorph 4.0.0 (Adams et al. 2021) using the procD.lm function over 1000 permutations with the Procrustes coordinates and centroid size. Furthermore, to establish whether there are significant differenc-es in body shape among species, a pairwise comparison test was performed using the pairwise function as implemented in library RRPP  over 1000 permutations.

Results
The pPCA indicated that the first three components explained 80% of the total variance (PC1: 52.6%; PC2: 17.6%; PC3: 11.6%). Species located on the positive axis of PC1 include T. irregularis, P. bulleri, and R. lentiginosus; O. heterospila, M. argenteus, C. pearsei, and K. ufermanni were located on the negative axis; and C. intermedium, V. hartwegi, and W. nourissati were located on the middle axis. The deformation grids showed variation among species on the positive axis related to decreased body height, elongation of the caudal peduncle, a convex base of the anal fin, and a narrow distal section. Species on the negative axis showed a deep body height, shortened caudal peduncle, and concave base of the anal fin. According to the morphotypes, the lotic species T. irregularis, P. bulleri, and R. lentiginosus were on the positive axis, while lentic species on the negative axis included O. heterospila, M. argenteus, C. pearsei, and K. ufermanni. However, the lotic species C. intermedium and W. nourissati, as well as the lentic species V. hartwegi, were in the middle of the axis ( Fig. 2A, B) In PC2, species found in the positive axis included C. pearsei, K. ufermanni, C. intermedium, T. irregularis, and V. hartwegi, while O. heterospila, W. nourissati, R. lentiginosus, M. argenteus, and P. bulleri were found on the negative axis ( Fig. 2A). The deformation grids showed that the most remarkable deformation occurred in the cephalic region. Species on the positive axis exhibited heads with straight profiles and mouths in a terminal position, while the eyes were displaced posteriorly and slightly enlarged. Species on the negative axis showed rounded heads and a ventral mouth position, while the eyes were both smaller in size and were displaced anteriorly ( Fig. 2A, 2B).
In PC3, W. nourissati was the most differentiated on the positive axis, exhibiting accentuated variation in the cephalic region with increased head size and a notable anteroventral displacement of the mouth. Additionally, the eyes and pectoral fins of W. nourissati were displaced posteriorly. On the negative axis, the remaining species were equally distributed with short heads, rounded profiles, and small mouths (Fig. 2B).
The dendrogram based on Mahalanobis distances showed that C. intermedium differed the most in body shape, followed by the C. pearsei and K. ufermanni groups. The remaining species formed two groups-one composed of P. bulleri, R. lentiginosus, and T. irregularis and another composed of M. argenteus and O. heterospi-la. The second most similar species were W. nourissati and V. hartwegi. The phylogenetic signal value of the Kmult statistic was 0.765, with a significance value of P = 0.308 and displaying no significant effect of phylogeny on body shape under the Brownian motion evolutionary model (Fig. 3).

Discussion
In the pPCA, species were placed in morphospace based on body length and depth, followed by variation in the cephalic region based on changes in head size and profile, mouth position, and eye position and size. Additionally, some morphological changes were evident based on the position and size of the fins. The variation observed in these morphological characteristics has been closely associated with the environments, locomotion, and feeding of fish (Wootton 1990;Muschick et al. 2012;Feilich 2016;Fugi unpublished * ). In African and South American cichlids, variation in these morphological characteristics has been fundamental in adaptive radiation events because it facilitates phenotypic and lineage diversification through the exploitation of ecological opportunities (Kocher et al. 1993;Cooper et al. 2010;Muschick et al. 2014;Arbour and López-Fernández 2016). The morphological body variation patterns observed in South American cichlids are similar to those observed in species of the Theraps-Paraneetroplus clade, which supports the hypothesis of Middle American cichlid diversification via ecological opportunity as proposed in previous works (Arbour and López-Fernández 2016;Feilich 2016;Říčan et al. 2016).
In the morphospace and similarity analysis, the most notable groupings were T. irregularis, R. lentiginosus, and P. bulleri, all of which presented elongated bodies and caudal peduncles as well as pelvic fins that were positioned ventrally. Ecomorphological studies have reported that these characteristics are functionally associated with high-velocity current environments (Lowe-McConnell 1991;Lauder and Tytell 2005;Pease et al. 2012;Feilich 2016). Elongated bodies are more hydrodynamic, while fins in the ventral position permit fish to maintain stability (Drucker et al. 2005;Lauder and Tytell 2005;Pease et al. 2012;Feilich 2016;Han et al. 2020). Soria-Barreto and Rodiles-Hernández (2008) reported the same morphological pattern for T. irregularis and R. lentiginosus in the Lacandon rainforest (Montes Azules Biosphere Reserve, Mexico), and Gómez-González et al. (2018) in V. hartwegi. Additionally, the mouth positions of the three species tend to be sub-terminal or ventral, which is associated with foraging for algae and invertebrates on the bottom, particularly on the surfaces of rocks with silt and sand substrates (Keast and Webb 1966;Miller et al. 2005; Artigas-Azas 2005b).
Wajpamheros nourissati, the outlier of the cichlids analyzed, is the only species that has long and thick lips. This is a characteristic associated with feeding between cracks, on rock surfaces, and on the substrate (Winemiller et al. 1995;Artigas-Azas 2005a;López-Fernández et al. 2014;Říčan et al. 2016). This characteristic has been reported in Middle and South American cichlids, which feed on benthic components and live in habitats with a variety of substrates including sand, silt, and organic matter in fine and coarse particles (Barlow and Munsey 1976;Moreira and Zuanon 2002;Hahn and Cunha 2005;Elmer et al. 2010;López-Fernández et al. 2012). Additionally, W. nourissati has well-developed pectoral fins that provide stability in habitats with currents of moderate velocity (Allgayer 1989). These morphological traits can be functionally important for exploiting specific habitats and alimentary resources while reducing competition with other sympatric cichlids.
The variation associated with the cephalic region revealed that the majority of species occupy a position in the morphospace that corresponds to their respective clades. For example, members of the Theraps clade (i.e., C. pearsei, C. intermedium, K. ufermanni, and T. irregularis) are positioned toward the superior part of the morphospace, while members of the Paraneetroplus clade (i.e., M. argenteus, O. heterospila, P. bulleri, and R. lentiginosus) are located toward the lower region. However, convergence between the two clades was also observed, largely in the variation of the cephalic characteristics of V. hartwegi and W. nourissati. This notably coincides with the variation of the cephalic characteristic in members of the opposite clade in both cases. Comparative studies have revealed patterns of rapid diversification between lineages and phenotypes through habitat-and diet-related morphological diversity (Arbour and López-Fernández 2016;Feilich 2016). Furthermore, it has been shown that the early radiation of certain Neotropical cichlid tribes in South America occurred rapidly, which resulted in a pattern of conflicting divergence (López-Fernández et al. 2013;Astudillo-Clavijo et al. 2015).
Evidence of the low phylogenetic signal of the body shape thus disproves the hypothesis of evolutionary non-independence. However, as the value of the phylogenetic signal can be determined by several processes and evolutionary rates (Revell et al. 2008), it raises the question of what specific causes determine body shape. In the evolution of quantitative traits, there is evidence that a low phylogenetic signal is related to speciation events caused by divergent selection and adaptive processes (Revell et al. 2008). In several Neotropical cichlid clades, this is the main cause for diversification events related to ecological opportunity (López-Fernández et al. 2012, 2013McMahan et al. 2015;Feilich 2016;Říčan et al. 2016).
In this context, some species of both clades (Theraps-Paraneetroplus) exhibited high convergence in the variation associated with body length and depth, fins position, and eye size and position. This is evident in C. intermedium, V. hartwegi, and W. nourissati, which showed similar body shapes despite being considered as different ecomorphotypes. The remaining species exhibited a morphological pattern consistent with what was expected from the corresponding habitat. The lentic species (C. pearsei, K. ufermanni, M. argenteus, and O. heterospila) showed a clear morphologically divergent pattern with respect to the lotic species (R. lentiginosus, T. irregularis, and P. bulleri). In the case of C. intermedium, V. hartwegi, and W. nourissati, it is probable that habitat preference is determined by other morphological traits that are likely linked to the cephalic region and associated with feeding behavior. This adaptive process has been documented in other fish groups; for example, in the Pomacentridae family, variations in cephalic traits explain feeding behavior and diet and are an important predictor of trophic habit (Aguilar-Medrano et al. 2011).
Based on the analysis of body shape and head characteristics, the lack of congruence in the order of species in morphospace does not fully support the ecomorphological classification described by Říčan et al. (2016). However, the results support the hypothesis of functional independence between the cephalic and postcranial regions identified for Middle American cichlids (Říčan et al. 2016). In regard to the cephalic region, the occurrence of five ecomorphotypes associated with feeding behavior was identified. Meanwhile, the postcranial region showed the lentic and lotic ecomorphotypes associated with their respective environments. In both instances, molecular phylogeny supported the recurrent evolution of diverse cephalic and postcranial ecomorphotypes among Middle American cichlids (Říčan et al. 2016). Additionally, modularity has been considered an evolutionary factor leading to patterns of variation among cichlids. Modularity studies in African cichlids have centered on modules associated with feeding: cichlid species that feed by suction have functional modules and those that feed by foraging have developmental modules (Parsons et al. 2012).
Species of the Theraps-Paraneetroplus clade show highly varied and convergent morphologies that are largely promoted by ecological opportunities associated with habitat and feeding preferences (Albertson and Kocher 2001;McKaye et al. 2002;Kassam et al. 2003;López-Fernández et al. 2013;Feilich 2016;Říčan et al. 2016). Body size and depth are features that appear unrelated to phylogenetic relations, whereas the head features of the majority of species exhibit patterns of variation associated with their phylogeny. However, further analyses of the shape, role, and evolution of these morphological attributes are necessary to understand their significance in the diversification of Middle American cichlids.

Conclusions
In this paper, the morphological variation of the Theraps-Paraneetroplus clade was divided into three groups within the morphospace. These variations are related to body length, body height, head shape, mouth position, and eye size and position. In particular, W. nourissati was the most divergent species due to its cranial characteristics, which are largely related to the shape of the head and the position of the mouth. Body size and height were the variables that best described the position of the species in the morphospace. This facilitates the recovery of lotic and lentic ecomorphotypes for seven species, with only C. intermedium, V. hartwegi, and W. nourissati showing incongruity. The presence of the phylogenetic signal disproves the non-independence hypothesis, but evidence suggests that body shape results from adaptive processes related to ecological opportunity. Although the results of the ANOVA with Procrustes distances and size of the centroid were contradictory, groupings in the morphospace and dendrogram were consistent with the ecomorphotypes and phylogeny. The pairwise comparison test showed statistical differences between all species, with the exception of V. hartwegi-P. bulleri. The morphological patterns found support the taxonomical validity of each species and can be used to describe body shape at the genus level. Future morphological evolution studies should consider cranial structures related to the capture and processing of food.