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Sexual dimorphism of Canthophrys gongota ( Teleostei : Cobitidae ) using landmark-based geometric morphometrics in the Atrai river of Bangladesh

Studies on sexual dimorphism of gongota Loach Canthophrys gongota (Local name: Pahari Gutum) was performed capturing them from the Atrai River of Dinajpur district in Bangladesh. Females had light blotches and patches with thick and rounded pectoral and pelvic fins while males having dark blotches and patches with thin and comparatively pointed paired fins. Body size, lengths of the anal fin and distances between the bases of pectoral, pelvic and caudal fins were significantly different (5.62 < F < 11.65, P ˂ 0.05) between the sexes of C. gongota. The expansion factors of mean thin-plate grids and vectors also showed that the head region of males was statistically different from females, whereas abdomen and tail of the females were considerably broader than those of the males. Both PCA (principal component analysis) and DFA (discriminant function analysis) plots showed morphologically little overlapping of landmark points which discriminated the females from the males. These findings are the first records on the sexual dimorphism of this rare species that would be baseline in a future study.


Introduction
Sexual dimorphism is the systematic variation in form and/or color between the sexes of animal taxa. Fishes display their sexual dimorphic traits in body size, shape, fins, and/or ornamentation (Echeverria, 1986). Morphological variations between sexes are not only found in primary sexual characters, but also in secondary sexual traits not directly associated with their spawning (Kitano et al., 2007). Some fishes exhibit temporary sexual characteristics (e.g. colors) but others show permanent (e.g., Clasper organ) structures (Saurabh et al., 2013). However, many fishes do not show any sexual disparities during their life cycle (Chu-Koo et al., 2009). Sexual dimorphism of fishes can be determined by the surveillance of fish gonads by dissecting the individuals regardless of maturity and breeding season. In fishes, some morphological traits differ between sexes apparently playing vital roles in their spawning period (Plongsesthee et al, 2012). Although sexual behaviors are the most crucial facts in reproduction and autecology of fishes but no available information present on these aspects for loaches (Bohlen, 2008). The key sexual dimorphic traits are commonly used for identification and taxonomy of loach fishes but also have been applied to classify genera and subgenera (Nalbant, 1994).
In Bangladesh, a total of 265 species of freshwater fishes is recorded where at least 24 species of loaches belonging to 10 genera of three families (Rahman, 2005). The Atrai River is one of the major rivers in Dinajpur district of Bangladesh (Islam and Mia, 2016;Islam et al., 2017;Islam et al., 2018) enriched with five species of freshwater loaches (Chaki et al., 2015). The gongota loach, Canthophrys gongota (Hamilton, They show no external sexual dimorphism (Siddiqui et al., 2007) and reported in the rivers close to foothills (Kottelat, 2012) from May to June (Saha and Saha, 2011). As some fishes primarily exhibit monomorphic traits but sexual dimorphic characters between sexes are anticipated because males and females show various reproductive roles for partner selection expressing their morphological variations (Casselman and Schulte-Hostedde, 2004). Besides, abdominal shape as a reproductive structure of mature fishes would be expected to change during spawning season (Hassell et al., 2012). Geometric morphometrics is a graphical approach that provides more facilities to reveal, illustrate and quantify the structural variations of animal taxa using a more significant approach (Adams et al., 2004;Slice, 2007). Now, it is widely applied to study the sexual dimorphism and ontogeny in fishes (Kitano et al., 2012) including their intraspecific phenotypic differences. Even though sexual distinctions between males and females have been studied on some loaches (Bohlen, 2008;Plongsesthee et al., 2012) but without any earlier record. However, null hypothesis is that there is no significant variations between two sexes of this loach. Therefore, an attempt was made to assess the sexual dimorphism of C. gongota with a view to identify their key morphometric traits that will be convenient to separate females from the males.

Study area
A total of 128 individuals (64 males and 64 females) of C. gongota were collected directly from fishermen of the Atrai River (25.924° N, 88.724° E) in Dinajpur district located in the northwest region of Bangladesh (Fig. 1). Fishermen caught fishes by using fine meshed seine net (15 × 3.5 m 2 , mesh size 4 mm). Fishes were immediately (within 1.5-2.0 h) transported with ice box and carried to the laboratory of the Department of Fisheries Biology and Genetics under Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh.

Identification and measurement of specimens
At first, external form and/or ornamentation of the collected specimens were examined to find out any traits as reliable indicators in their body followed by Plongsesthee et al.  (Table I; Fig. 2 and 3). Finally, fish samples were dissected to collect their gonads for precision of prediction by visual observation of sexes. After gonad collection, sexes were finally detected by observing their testes and ovary (Esmaeili et al., 2017).
Before dissection, photographs of the specimen were taken in order to grasp their main sexual dimorphic characters using geometric morphometric analysis. Individuals that had damaged fins (e.g. dorsal and caudal fins) not considered for imaging and analysis.

Digitalization of images and landmarks
During photography, each specimen placed in the center of a dissecting pad (32 × 23 cm 2 ) with a left side of its body facing. A total of 64 males and 64 females were taken into consideration for their photographs (4000 × 3000 pixels, 180 dpi) using a digital camera (Canon PowerShot SX510 HS) where fins of each fish were spread out and pinned in place to offer a distinct view of insertion points into their body. For measuring, a ruler was placed against the lower edge of dissecting pad. Twenty-four landmarks (equal to 24 x and 24 y coordinates) were selected to analyze body shape differences between the sexes using landmark-based geometric morphometrics. Pins inserted on each specimen at all landmarks before taking photographs to make sure the accuracy of landmark locations during digitization. Multiple photographs of an individual were taken to check the acquisition of a quality image. After photography, images reviewed to select the best image for each specimen. Landmark clarity, lighting and angle were examined when selecting a photograph. Once chosen, photograph file was renamed with specimen identity number to classify them accurately as their sex (Santos and Quilang, 2012;Reiss and Grothues, 2015). All pooled images were processed for digitization (Fig. 2) to decide sexual dimorphism where landmarks were digitized using tpsDig2 software (Rohlf, 2006). After opening the images through this software, the ruler was set for measuring distance and then mouse point was set on two landmark points to get correct and reliable measurements.

Statistical analysis
More or less mature fishes were used to lessen the effects of intrapopulation shape variations because of allometric growth pattern (Dorado et al., 2012). Specimen data put in rows and two-dimensional landmarks (x and y coordinates) along each row for general procrustes alignment (GPA) by rotating and translating to major axis for linear space (Rohlf and Slice, 1990). The resulting procustes distances were used to calculate in Multivariate Analysis of Variance (MANOVA) to observe whether the differences in morphological traits between sexes of C. gongota are significant or not. The analysis of similarity (ANOSIM) is a hypothetical test that was used to evaluate the phenotypic variations between sexes of this loach. The value of 'R' was calculated as 4 (r b -r w )/N(N-1), where 'r b , is the average rank of all distances between groups, 'r w ' mean rank of all distances within groups and 'N' is the total number of sample (Clarke, 1993). The thin-plate spline algorithm was used to calculate the mean body shape between sexes of C. gongota (Bookstein, 1991). Discriminant Function Analysis (DFA) was also used to evaluate the total amount of difference between males and females. Principal Component Analysis (PCA) scatter diagram was also used to visualize the degree of differences between the sexes of this species.

Sexual dimorphism through general observation
In this study, it was observed that females had a larger body (standard length), broader abdomen, comparatively thick and rounded paired fins (pectoral and pelvic) having relatively greater distances between bases of two fins than males ( Fig. 3  and 4). In contrast, males had thin and slightly pointed pectoral and pelvic fins compared to the females. Besides, females also had reddish orange (during spawning period) to cream (in pre and post-spawning time) colored ovaries with the rough surface while males carried whitish colored smooth testes. Moreover, body size (standard length and body weight) and distances between bases of paired (pectoral and pelvic) and unpaired (anal and caudal) fins were significantly varied (F > 5.62, P ˂ 0.01) while no statistical divergences (F < 1.14, P > 0.05) were observed in the lengths of dorsal, pectoral, pelvic and caudal fins between sexes of C. gongota (Table I).

Sexual size dimorphism using geometric morphology
During an experiment, a significant difference (P ˂ 0.01) was observed between sexes of C. gongota based on body shape variations using MANOVA test (Wilk's lambda = 0.18, P ˂ 0.01) representing sexual dimorphism. The ANOSIM compared the mean of ranked dissimilarities between groups to mean of ranked dissimilarities within groups exhibiting variations (R = 0.61, P < 0.01) between males and females (Table III). This deformation grid depicted the shape transformation associated with first canonical variate (CV 1 ) axis where eigen value, % of variance and Mahalanobis distances between group were 1.77, 99.22% and 2.64, respectively discriminating the sexes. The deformation grid associated with CV 1 illustrated that the most effective discriminators were 1, 2, 4, 23 (head region); 6, 7 (dorsal fin); 10, 11 (caudal fin); 21, 22 (bases of pectoral fin); 17, 20 (bases of pelvic fin) and 13, 16 (anal fin bases) between the sexes of this loach (Fig. 5). Besides, higher values of expansion factor were found in the head region for males (> 1.17) and in abdomen mainly in front of pelvic fin bases for females (> 0.58).
Using principal components analysis (PCA), the variance of PCA 1 and PCA 2 were 79.68% and 8.23%, respectively. The points that cluster together had similar shapes but far removed showed different shapes (Fig. 6). A discriminant function analysis (DFA) was used to judge distinctions between males and females of C. gongota where the group was correctly classified as 97.66% (F = 7.01, P ˂ 0.01). The low overlap and high spread of DFA values explained higher and significant variations along mean values of DFA. The first six relative warps scores (RWs) of Pahari Gutum were RW 1 (36.22%), RW 2 (24.42 %), RW 3 (9.38%), RW 4 (6.87%), RW 5 (3.54%) and RW 6 (2.41%) accounted to 82.85% of total variance and eigen value of these scores were 0.077, 0.052, 0.0201, 0.015, 0.008 and 0.005, respectively. The percentage of RW 1 was larger showing higher differences compared to other RW scores. The first two relative warps accounted for 60.64% of total variance in 24 landmark points. Positioning in the plot relative to other individuals indicated the degree of similarity where individuals with positive amplitude on the first relative warp were similar in body shape and vice-versa. Body shape variations between sexes i.e. sexual dimorphism is the outputs of genetic changes mainly controlled by natural selection, choice of a couple and ecological selection (Blanckenhorn, 2005;Hassell et al., 2012) that were not considered in this study. This study reported that females of C. gongota were significantly larger in size than males which were consistent with the morphology of Cobitis elongatoides and Sabanejewia balcanica (Bohlen et al., 2008), C. calderoni (Valladolid and Przybylski, 2008) and Lepidocephalichthys (Havird et al., 2010). The reasons may be that female of loach may convert their energy for growth rather than reproduction until the second year whereas male uses more energy for breeding from their first-year life cycle (Eros, 2003). Studies regarding sexual dimorphism of this species have focused only on age and growth differences between males and females (Talwar and Jhingran, 1991). It was also observed that females had large and extended abdomen than those of males may be due to larger gonads demonstration which is similar to a loach L. goalparensis (Das et al, 2012). The males of Schistura sexcauda have a more slender abdomen than females (Plongsesthee et al., 2012). Females of this gongota loach had a series of light brown blotches along sides and a less dark patch beneath the eyes compared to males which may possibly work as a functional gesture to entice females for mating or to counsel against males. Kottelat (2012) studied on over 60 loach species where all fishes carry small and extended body shape with different colored ornamentations, spots, and blotches. A sexually dimorphic black blotch present in pectoral fins of males S. aurantiaca, S. cf. aurantiaca and S. sexcuada while it is absent in females (Plongsesthee et al., 2012). In common, males have statistically larger pectoral fins than females in most loaches such as C. jadovaensis (Mustafię et al., 2008) and S. cf. aurantiaca (Plongsesthee et al., 2012). However, there were no significant variations in the lengths of pectoral, pelvic, caudal and dorsal fins between sexes of C. gongota which is similar to the morphology of S. mahnerti (Plongsesthee et al., 2012). But, a significantly larger anal fin was recorded in females than in males of this loach. Moreover, females of C. gongota also had significantly greater distances between the bases of pectoral, pelvic and caudal fins compared to males.
Landmark-based geometric morphometric is an important tool to examine the taxonomic and morphometric distinctions within and between species (AnvariFar et al., 2011;Vilizzi and Kováč, 2014). There are no previous findings on sexual dimorphism of C. gongota (Pahari Gutum) or any other loaches (except three species of Cobitis reported by Mousavi-Sabet and Anvarifar, 2013) using truss-networks based geometric morphometric analysis. Therefore, it is not possible to compare the contemporaneous results with previous ones. However, the males and females of C. gongota showed sexual dimorphism based on their size independent morphometric traits which were similar to Cobitis sp. (Buj, et al., 2014) and to eight-barbel loach Lefua sp. (Aoyama, 2007). Using ANOSIM analysis, highly significant phenotypic variations also existed in the external morphology between sexes of C. gongota which may due to different roles played during their reproduction. These phenotypic changes may be due to genetical, environmental or their combinations (Pinheiro et al., 2005). Thin plate analysis between males and females of gongota loach represented that variations were higher in the head and abdominal region compared to other body regions. Moreover, landmark plots presented low overlaps of shapes in both sexes of C. gongota where main dissimilarities mainly on the head and abdominal regions (an area between pectoral and pelvic fin bases). In Mesopristes cancellatus, differences were observed in the abdominal area and minimal variation on the tail between males and females (Barazona et al., 2015). The PCA scatter plot of C. gongota showed little overlapping of plots which implied that landmarks represented significant differences in body shapes of two sexes. In this study, PCA plots presented that both sexes of this fish population converge or overlapped at low level along the horizontal axis showing higher isolation between the sexes. Discriminant function analysis (DFA) could be a useful method to distinguish different species of the same genus or different stocks of the same species with respect to stock management programs (Karakousis et al., 1991). The overlap and spread of DFA represented 97.66% classification for C. gongota while 58.10%, 64.70% and 44.10 for Cobitis sp., C. faridpaki and C. keyvani, respectively (Mousavi-Sabet and Anvarifar, 2013). Besides, the individuals also grouped into 95.80% for Salmo salar (Solem and Berg, 2011) as different taxa.
However, variations between males and females of a fish species are important tools for fish stock assessment. The landmark-based geometric morphometric approach enabled us to quantify and visualize morphometric differences between sexes of C. gongota. This finding will be useful to identify sexes of C. gongota species where alteration patterns of sexes can be supportive to both in rearing and culturing with other developmental aspects of this species. Lastly, this landmark-based geometric morphometric study would be a baseline for future study on loaches within and among populations.