New Insights into the Male Morphotypes of the Amphidromous Shrimp Macrobrachium olfersii (Weigmann, 1836) (Caridea: Palaemonidae) and a Discussion on Social Dominance Hierarchies

© 2022 Academia Sinica, Taiwan Open Access New Insights into the Male Morphotypes of the Amphidromous Shrimp Macrobrachium olfersii (Weigmann, 1836) (Caridea: Palaemonidae) and a Discussion on Social Dominance Hierarchies Rafael Carvalho Santos1,* , Caio Santos Nogueira1, Milena Silva Jaconis1, Thiago Maia Davanso1, Rogerio Caetano Costa1, and Gustavo Luis Hirose2 1Laboratory of Biology of Marine and Freshwater Shrimps (LABCAM), Department of Biological Sciences, Faculty of Sciences, São Paulo State University (UNESP), Bauru, São Paulo, Brazil. *Correspondence: E-mail: [email protected] (Santos). [email protected] (Nogueira); [email protected] (Jaconis); [email protected] (Davanso); [email protected] (Costa) 2Laboratory of Carcinology, Department of Biology, Federal University of Sergipe (UFS), São Cristóvão, Sergipe, Brazil. E-mail: [email protected] (Hirose) Received 30 April 2022 / Accepted 7 October 2022 / Published 26 December 2022 Communicated by Benny K.K. Chan Male morphotypes in a population may lead to the development of social dominance hierarchies in crustacean species. Currently, Macrobrachium is the decapod crustacean genus with the largest record of species that present the development of hierarchies. Macrobrachium olfersii has morphological characteristics that indicate the presence of male social dominance within its populations. Thus, the present study tested the hypothesis of the occurrence of male morphotypes in M. olfersii through morphometric and morphological analysis of the chelipeds. Sampling was carried out from March 2018 to October 2021 in seven points along the Jequitinhonha River, Northeast Brazil. A total of 264 males were collected with carapace length (CL) ranging from 4.01 to 23.70 mm. Morphological sexual maturity size was estimated at 8.95 mm CL. The morphometric and morphological analysis confirmed the presence of three adult male morphotypes: M1, M2, and M3. The characterization of the different morphotypes was mainly due to the variation in size, shape, and morphology of the largest cheliped of the second pair of pereopods. Most morphometric relationships differed significantly (p < 0.01) among the three morphotypes, mainly between M3 against M1 and M2. The variation in the propodus shape was also evident. This trait and the angulation of the spines differed significantly between morphotypes (p < 0.01), with the propodus of morphotype M3 being more robust and carrying a greater number of spines than the others. The occurrence of social dominance and the exaggerated development of a cheliped (weapon) can be advantageous for dominant individuals when they need to compete for resources. This morphological trait can provide these individuals with advantages during fights and guarantee access to the best resources, whether they are shelter, food, or sexual partners. Our results add new information to the biology of M. olfersii, as well as the genus Macrobrachium, and the occurrence of social dominance in species of this group. In addition, by describing these morphotypes in detail, using a set of complementary morphological and morphometric techniques, it is possible to access the differential morphology along the M. olfersii males, as well as confirm a life history trait found in several Macrobrachium species. Key words: Allometry, Chelipeds, Geometric morphometrics, Relative growth, Decapoda. Citation: Santos RC, Nogueira CS, Jaconis MS, Davanso TM, Costa RC, Hirose G. 2022. New insights into the male morphotypes of the amphidromous shrimp Macrobrachium olfersii (Wiegmann, 1836) (Caridea: Palaemonidae) and a discussion on social dominance hierarchies. Zool Stud 61:83. doi:10.6620/ZS.2022.61-83. Zoological Studies 61:83 (2022) doi:10.6620/ZS.2022.61-83 1 © 2022 Academia Sinica, Taiwan BACKGROUND Social dominance and hierarchies are mechanisms that provide access for individuals of several groups of invertebrates to better resources (i.e., shelter, food, sexual partners) (Dugatkin and Dugatkin 2007; Stewart and Tabak 2011; Soundarapandian et al. 2013; Lord et al. 2021). When this mechanism is associated with competition between males of the same population, a polymorphism is commonly observed (Soundarapandian et al. 2013). The process of becoming a dominant individual within a population requires a high initial energy investment (López and Martín 2001; Karplus and Barki 2019; Lord et al. 2021). However, there is a compensatory return since dominant individuals are less confronted, minimizing the energy spent on agonistic events (López and Martín 2001; Lord et al. 2021). Among invertebrates, there are numerous records about the establishment of social dominance hierarchies in species of dragonflies, cephalopods, water bugs, and spiders (Campanella 1975; Ahtiainen et al. 2006; Boal 2006; Pérez et al. 2019). However, in crustaceans, this feature also is common, especially in infraorders of decapods (Winston and Jacobson 1978; Stewart and Tabak 2011; Karplus and Barki 2019; Lord et al. 2021). In decapod crustaceans, males develop a differential morphology of their chelipeds to become dominant (Karplus and Barki 2019). These structures are used as weapons in agonistic events, influencing social hierarchies (i.e., dominant morphotypes and submissive morphotypes) within a population (Kuris et al. 1987; Mariappan et al. 2000; Correa et al. 2003; Karplus and Barki 2019; Hamasaki and Dan 2021). Among decapods, morphotypes have been described in brachyuran (Laufer and Ahl 1995; Sal Moyano and Gavio 2012), anomurans crabs (Bueno and Shimizu 2009; Takano et al. 2016), freshwater crayfishes (Hamasaki et al. 2020), and several genera of caridean shrimps (Thiel et al. 2010; Bauer et al. 2014; Karplus and Barki 2019). Among caridean shrimps, Macrobrachium Spence Bate, 1868 currently encompasses the largest number of species that have male morphotypes. Male morphotypes have been so far described for the species Macrobrachium acanthurus (Weigman, 1836) by Rios et al. (2021), M. amazonicum (Heller, 1862) by MoraesRiodales and Valenti (2004), M. brasiliense (Heller, 1862) by Nogueira et al. (2020), M. grandimanus (Randall, 1840) by Whortam and Maurik (2012), M. idella (Hilgendorf, 1898) by Soundarapandian et al. (2013), M. rosenbergii (de Mann, 1879) by Kuris et al. (1987), and M. tenellum (Smith, 1871) by VargasCeballos et al. (2021). Recently, molecular phylogeny data revealed that the genus Cryphiops Dana, 1852 is a junior synonym within Macrobrachium (Mantelatto et al. 2021). Thus, the species Cryphiops caementarius (Molina, 1782), which has different male morphotypes described (Rojas et al. 2012), was added to the total number of Macrobrachium species that show this type of social dominance. The occurrence of male morphotypes in Macrobrachium olfersii (Wiegmann, 1836) is a feature that has been historically discussed. This was suggested when molecular and morphological analysis indicated that two other species of Macrobrachium (M. birai Lobão, Melo & Fernandes, 1986 and M. holthuisi Genofre & Lobão, 1978) were a junior synonym of M. olfersii (Pileggi and Mantelatto 2010 2012). One of the main morphological characteristics used to separate these three species was the morphology of the second pair of chelipeds, precisely the structure that presents the greatest morphological variation among male morphotypes. The authors argued that this variation was not due to interspecific differences but to the possible existence of male morphotypes in this species (Pileggi and Mantelatto 2010). By then, the existence of morphotypes for M. olfersii still required confirmation. The population structure of M. olfersii supports the existence of morphotypes in this species. Previous studies have shown that only males reach the maximum observed sizes for this species, so males are mainly grouped in larger size classes in relation to females (Lombardi et al. 1996; Pescinelli et al. 2016). This pattern of body size difference was observed in studies that addressed populations before and after the taxonomic revision involving the M. olfersii species complex (Pescinelli et al. 2016). In addition to the variation in body size between males and females, there is also an evident difference in the size of the second pair of chelipeds between these groups; males have an exaggeratedly more developed cheliped than females and a more pronounced heterochelic pattern (Ammar et al. 2001; Mossolin and Bueno 2003; Pescinelli et al. 2016; Müller et al. 2018). However, none of these previous studies investigated the existence of different morphotypes in males. Recently, a study explored the occurrence of male morphotypes in M. olfersii using specimens from different populations that occur along the Brazilian coast (Rossi et al. 2022), although, apparently this study did not sample all size classes of males of M. olfersii. Furthermore, when using specimens from different populations, possible morphological and morphometric differences could also bias the correct identification of polymorphic groups. Thus, morphological, morphometric and behavioral studies carried out with individuals from the same population are still necessary in order to corroborate the existing previous information. page 2 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan Analyzing morphometric relationships between the growth of different body structures is fundamental to determining hierarchical groups in a population, since differences in allometric coefficients can influence ontogenetic development and consequently the size and shape of structures (Hartnoll 1974; Rosenberg 2002; Klingenberg 2016). The evaluation of the shape (assessed via geometric morphometrics) of the cheliped can contribute to the discrimination of male morphotypes in shrimp species, despite not being commonly used for this purpose (Nogueira et al. 2022a). In most cases, discrimination only uses the relative growth analysis (linear morphometrics) (Kuris et al. 1987; Moraes-Riodades and Valenti 2004; Rojas et al. 2012; Wortham and Maurik 2012; Pantaleão et al. 2014; Nogueira et al. 2020; Rios et al. 2021). Given the evidence that points to high variability in morphology among males of M. olfersii, the present study aims to test whether there is a presence of male morphotypes in this species using a single population. Different morphometric and morphological aspects were evaluated between adult males to determine the variation in size, relative growth, shape, and ornamentation of chelipeds. We predicted that if there were different male morphotypes, they should present evident differences between the evaluated morphometric and morphological aspects. This pattern should follow the one observed in other Macrobrachium species, with dominant morphotypes investing more in the development of chelipeds, therefore presenting overdeveloped claws. MATERIALS AND METHODS Sampling Sampling was carried out in the Jequitinhonha River, Bahia, Brazil (15°58'5.941"S, 39°35'11.983"W). We conducted ten campaigns: March 2018, August 2018, June 2019, October 2019, February 2020, August 2020, November 2020, April 2021, July 2021, and October 2021. In each campaign, seven points were sampled along the river, using two different sampling methods. These methods were (1) a cylindrical trap with a rectangular mesh (mesh openings 1 mm wide and 5 mm long, base with 36 cm in diameter and 60 cm in height) that was left for four hours; and (2) a handdrawn trawl net (3 m long, 1.80 m high, 10 mm mesh), which was thrown five times in each point, covering a perimeter of approximately 15 meters per point. These two methodologies were applied in an effort to increase the range of individuals captured. The expectation was to find differences among the sampling methods, with the cylindrical traps capturing more of the larger individuals, and the hand-drawn trawl capturing a wider range of sizes as well as a higher number of individuals, due the low selectivity of this sampling method (Polet 2000). All shrimps collected at the sampling site were sorted into plastic bags (containing local water) according to the sampling point and collection method. The shrimps were then transported to the laboratory. Individuals were identified at the species level using specific literature (Melo 2003) and separated by sex through the presence (males) or absence (females) of appendix masculina in the endopod of the second pair of pleopods (Valenti et al. 1987). Individuals of M. olfersii from the seven sampling points were considered as a single population due to reproductive characteristics of the species (amphidromous) that involve migration of individuals along the course of the river and the lack of geographic barriers. Measurements Males of M. olfersii were measured for carapace length (CL), ischium (IL), merus (ML), carpus (CaL), propodus (PrL), dactylus (DL), total length of the chelipeds (ChL) of the second pair of pereopods (Fig. 1A and B), and propodus’ height (PrH) using a digital caliper (accuracy 0.01 mm). The structures’ length consist of the measurement from the article base to the posterior region of the same article, meanwhile the propodus height consists of the larger distance in the palm. The ChL corresponds to the sum of the length of all the articles (ischium, merus, carpus, propodus). Measurements were taken from both chelipeds of the second pair of pereopods. Individuals that presented any type of injury on the articles that constitute the chelipeds, that is, any missing body parts in the cheliped (major or minor) or one single article (such as the propodus) were excluded from the analyses. Before morphometric analysis, the presence of outliers was verified using the interquartile range method. When identified, outliers were removed from the dataset (Hawkins 1980; Knorr and Ng 1998). The normality of the data was tested using the Shapiro-Wilk test (α = 0.05) and the appropriate analyses were applied according to the parametricity of the data. To verify if the second pair of pereopods presented heterochely and handedness, a Mann-Whitney test was applied to each (α = 0.05), since these two characteristics can influence the morphometric relationships. For the heterochely, we applied the size of the cheliped (larger and smaller) as the independent variable, and for the handedness, we applied the side of the larger cheliped (right and left). For both analyses, measurements of the total length of page 3 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan Fig. 1. (A) Carapace of Macrobrachium olfersii (Wiegmann, 1836). Dimension of carapace length (CL) measurements. (B) Major cheliped of Macrobrachium olfersii. Exemplification of the dimensions used to measure the length and height of the articles of the larger cheliped. The same measurements were used for the smaller cheliped. (C) Propodus of the larger cheliped of Macrobrachium olfersii in the standard position used in the geometric morphometric analyses. Red and blue circles are the landmarks and semilandmarks, respectively. CL = Carapace length; IL = Ischium length; ML = Merus length; CaL = Carpus length; PrL = Propodus length; DL = Dactylus length; PrH = Propodus height. page 4 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan the chelipeds were used as dependent variables. Then the analyses were performed using Statistica Statsoft 7.0 software. Morphometric analysis Individuals were separated and grouped into possible morphotypes based on the observation of cheliped morphology, according to variations that configure a polymorphism among males (cheliped size, number, size and angle of spines, presence of setae, and pubescence in the propodus, and degree of heterochely) (Kuris et al. 1987; Moraes-Riodades and Valenti 2004; Nogueira et al. 2020; Rios et al. 2021). The previously established morphological categories were submitted to a principal component analysis (PCA), an exploratory analysis that delimits the formation of groups based on a matrix containing the morphological variables measured to determine which variables are the most significant in the definition of groups. A non-hierarchical K-means cluster analysis (Sokal and Rohlf 1979) was applied to the morphometric data to initially separate juveniles from adults and then the possible morphotypes within adults. K-means is based on previously established groups through an iterative process and aims to minimize the variance within the groups and maximize the variance between the different groups. The results of the K-means age groups were refined by discriminant analysis (α = 0.05). Discriminant analysis (DA) was then applied to verify significance, refine data, and assess the classification of the previously defined groups (by initial morphological analysis), establishing the final division of the morphological groups (Sampedro et al. 1999). Both analyses were performed using the PAST 4.05 software. The validated groups (juveniles and morphotypes) were compared by analyzing the relative growth of the articles that constitute the chelipeds. This analysis was performed by linear regression (α = 0.05). Then, an analysis of covariance (ANCOVA, α = 0.05) was applied to verify if there were differences between the angular or linear coefficients of the morphometric variables between groups, as well as if the data of each morphological group were better adjusted to a single linear equation or if they must be represented by different linear equation (Pantaleão et al. 2014). Relative growth is a method that assesses the relationship between different body dimensions (dependent variables) with an independent variable (CL) (MoraesRiodades and Valenti 2002). This analysis is based on the allometric equation y = a.xb (Hartnoll 1978), which was linearized by the logarithmic equation lny = lna + b.lnx, where y = the measured dependent variable, x = the independent variable (CL), a = the point at which the line fixes on the coordinate axis (intercept), and b = the curve representing the allometric coefficient of the structure (slope). The allometric constant values were evaluated using Student’s t-test (α = 0.05), using the Statistica Statsoft 7.0 software. The null hypothesis H0: b = 1, would indicate allometric status as positive allometry (b > 1), negative allometry (b < 1) and isometry (b = 1) (Hartnoll 1978). Size at the onset of sexual maturity The result of the most explanatory variables of PCA used to separate age categories (juveniles and adults) was applied to estimate the size at the onset maturity (SOM). SOM was estimated using the CL50% method (Sampedro et al. 1999). To estimate the maturity value, individuals were separated into size classes, based on carapace length (CL; independent variable) using the Sturges formula, and according to the relative frequency of each class (dependent variable). Then, the data were fitted to the logistic curve (y = 1 1+er(CL-CL50)), with CL50 being the carapace length at which 50% of the population is mature and r the slope of the curve. Morphological analysis Morphological analysis was performed to describe the morphology of adult male morphotypes confirmed by morphometric analyses. To this end, the ornamentations (the same used for the initial separation of the groups), and the variation in the size and shape of the larger cheliped were described. The angulation of the spines present in the propodus of the male morphotypes was evaluated to observe if there was a significant variation in the angle of projection of these structures between the morphotypes since these are considered important morphological traits in the determination of male morphotypes in some species of Macrobrachium. Dominant morphotypes are expected to have spines distributed along the cheliped, with wider angulations in comparison to the subordinate morphotypes, suggesting that the spines work as defensive corporal features to the dominant morphotypes (Kuris et al. 1987; MoraesRiodades and Valenti 2004; Nogueira et al. 2020; Rios et al. 2021). We randomly analyzed 10 spines present on the propodus of the largest cheliped from 10 individuals of each determined morphotype (adults only). Photographs of the cheliped spines were taken using a stereomicroscope trinocular Zeiss Stemi 2000C. The angulation projection was measured using the angle tool from the software Zeiss AxioVision. After the measurements, the values of the spine angles were page 5 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan compared between the groups using a Kruskal-Wallis analysis (α = 0.05) to verify if there was a difference between them, followed by a posteriori Dunn test (α = 0.05). Geometric morphometrics (General procedures) To confirm the presence of morphotypes, a geometric morphometrics tool was used to assess any statistical difference in the observed variation in the shape of the propodus of the largest cheliped of the morphotypes of M. olfersii. The images used in the geometric morphometric analysis were captured using a professional camera (Canon EOS Rebel T100) with an attached macro photography lens (100 mm). The same person photographed all specimens, and images were taken at the maximum resolution with a camera attached to a tripod. The distance between the lens and the structure was standardized in all photographs (40 cm). The constancy of the zoom and the position of the body structure were also standardized. In this step, only one structure was analyzed between each morphotype: the propodus of the largest cheliped of the second pair of pereopods. The positioning of the structures for photography was defined based on the handedness pattern between chelipeds. If there was no laterality, the largest propodus on both the right and left sides of the body could be considered analogous (i.e., symmetrically corresponding). Therefore, the photographs of the propodus were mirrored during the analysis so that they were oriented and standardized in the same anatomical position (Klingenberg et al. 2002). The propodus was analyzed because it is the main structure among the articles that constitute the second pair of chelipeds and the structure where the most notable morphological changes occur regarding sexual dimorphism, ontogenetic variation, or social dominance (Mariappan et al. 2000; Dennenmoser and Christy 2013; Lezcano et al. 2015; Karplus and Barki 2019). Landmarks and semilandmarks were digitized on the photographs to acquire the propodus shape. Eight landmarks were used to characterize the propodus shape and another 12 semilandmarks to capture the variation in the contour of the same structure (Fig. 1). All landmarks and semilandmarks were digitized using the tpsDig2 software (Rohlf 2005). A Generalized Procrustes Analysis (GPA) was then performed to superimpose, scale and rotate all landmarks and semilandmarks that were digitized in each structure (Rohlf and Slice 1990; Rohlf 2015). Errors related to measurements, capture of photographs, and digitization of landmarks were evaluated following the protocol proposed by Viscosi and Cardini (2011), which means performing the entire process twice. The weight matrix (partial warps + uniform components) that describes the shape of the structures was observed using the software tpsRelw v.1.49 (Rohlf 2010), while the variation of the shape of the structures was obtained using the software tpsRegr v.1.31 (Rohlf 2009). The size of the structures was estimated by the centroid size. This variable is defined by the square root of the sum of the squared distances of each landmark and semilandmark and the central mass of the structure (Bookstein 1997). It was also obtained by the tpsRelw v.1.49 software. Multivariate regression was performed between centroid size and shape variables to verify the presence of an allometric effect in the dataset. Residues generated by this regression were used in subsequent statistical analyses between male morphotypes of M. olfersii to remove the allometric effect from the dataset (Klingenberg 1998 2016). Propodus shape variation between male morphotypes was investigated by a multivariate analysis of variance (MANOVA) and a canonical variance analysis (CVA) using the residuals of the weight matrix (partial warps + uniform components). CVA was performed to explore the separation of morphotypes according to the propodus shape variation. Before performing these analyzes (MANOVA and CVA), a principal component analysis (PCA) was performed using the residuals of the weight matrix to check how many components represent more than 99% of the shape variation. Once the components were identified, they were used to run MANOVA and CVA. This method is commonly used to reduce the dimensionality of the data matrix and increase the power of the statistical test without affecting the representation of the variation in the shape of the structures (Mitteroecker and Gunz 2009). PAST v.1.8 software was used to perform MANOVA, CVA, and PCA. RESULTS A total of 264 males of M. olfersii were collected. The carapace size ranged from 4.01 to 23.70 mm. Chelipeds size of larger and smaller chelipeds was statistically different (U = 20347.50, p < 0.01), indicating the occurrence of heterochely. No pattern of handedness was observed (U = 20391.50, p = 0.85). Only the largest cheliped of each individual was used for morphology and morphometric analyses. PCA results corroborate the separation of M. olfersii males into juvenile and three different adult morphotypes (Fig. 2), designated as morphotypes 1, 2, and 3 (M1, M2, and M3, respectively). Principal components 1 and 2 (PC1 and PC2) explain 97.94% and page 6 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan 1.18% of the morphometric data matrix, respectively, accounting for 99.12% of the total explanation (Table 1). ChL followed by PrL were the structures with the greatest contribution to PC1, indicating these structures could be used to separate the groups. Morphometric analysis The separation of male morphotypes was confirmed by the discriminant analysis (p < 0.05), with more than 90% of the individuals correctly classified (J vs M1 = 99.17%; M1 vs M2 = 92.26%; M2 vs M3 = 97.71%). Among the 264 males of M. olfersii, 50 were identified as juveniles, 83 as M1, 98 as M2, and 33 as M3. The presence of outliers was not detected in the morphometric analysis. The results of the covariance analysis demonstrate that the relative growth of all morphometric relationships of the largest cheliped is statistically different between groups (Table 2), with all groups differing in the intercept of the line on the axis. There was an exception for the relationship PrH vs CL between juveniles and M1, which differed in the slope of the line. Despite the significant difference, relative growth demonstrates overlap in the size classes between Fig. 2. Macrobrachium olfersii (Wiegmann, 1836). Principal Component Analysis (PCA) of morphometric variables. Values indicate the projection of components 1 and 2 (PC1 and PC2). Table 1. Macrobrachium olfersii (Wiegmann, 1836). Correlation (Cor.) and contribution (Con.) values of the morphometric variables resulting from the Principal Component Analysis Variable PC1 PC2 PC3 PC4 Cor. Con. Cor. Con. Cor. Con. Cor. Con. CL 0.875 0.183 -0.480 -0.915 0.040 0.121 -0.011 -0.049 IL 0.910 0.077 -0.295 -0.227 -0.100 -0.123 0.037 0.069 ML 0.991 0.162 0.024 0.036 -0.025 -0.059 0.098 0.353 CaL 0.988 0.172 0.007 0.011 -0.074 -0.188 0.098 0.374 PrL 0.993 0.414 0.068 0.257 0.071 0.432 -0.060 -0.554 DL 0.960 0.197 -0.040 -0.075 -0.243 -0.730 -0.126 -0.571 PrH 0.933 0.126 0.149 0.183 -0.231 -0.456 0.065 0.192 ChL 1.000 0.824 0.010 0.076 0.005 0.063 0.013 0.243 Eigenvalue 255.78 3.08 1.19 0.52 % variation 97.94 1.18 0.45 0.20 CL = carapace length; IL = ischium length; ML = merus length; CaL = carpus length; PrL = propodus length; DL = dactylus length; PrH = propodus height; ChL = major cheliped length. Note: The numbers in bold correspond to the extremes of weighting for individuals in PC1. page 7 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan groups. Some individuals that have similar carapace length, however, differ in the cheliped length (Fig. 3). Linear regression analysis showed that all morphometric relationships differed between groups (p < 0.05). None of the morphometric relationships showed positive allometry. Negative allometries were found for all morphometric relationships referring to individuals from M1 and for the majority regarding juveniles and M2. There were exceptions for the PrH vs CL relationship in juveniles and the IL vs CL, DL vs CL, and PrH vs CL relationships for M2, which showed isometry. For all morphometric relationships of M3, isometries were found (Table 3). Morphological sexual maturity was measured from the ratio of juveniles and individuals of the M1 morphotype. The size at which 50% of the population reaches sexual maturity was 8.95 mm (Fig. 4), with the smallest adult male having 7.03 mm CL and the largest juvenile male having 11.47 mm CL. Morphological analysis The carapace length of juveniles ranged from 4.01–11.47 mm CL, with the largest cheliped length ranging from 8.60–18.04 mm ChL. The three morphotypes had a carapace length range of 7.03–13.70 mm for M1, 7.80–20.50 mm for M2, and 9.10–23.70 mm for M3. Regarding the largest cheliped length, the variation among the three morphotypes was 17.62–30.50 mm for M1, 24.51–54.50 mm for M2, and 32.30–81.60 mm for M3. This indicates that the cheliped size presents greater variability between morphotypes than CL. Table 4 shows the size variation of all structures referring to the largest cheliped among the male groups. Through the morphological differences observed in the largest cheliped, it was possible to separate three morphotypes of adult males (M1, M2, and M3). They varied in shape, size, presence of spines, and pubescence in the palm of the propodus. M1 individuals have a cheliped similar to juveniles, being relatively small, with short spines and slightly projecting on the upper surface of the propodus and carpus; there is no gap formation between the fixed finger and the movable finger. Although they do not show pubescence in the palm of the propodus, they present evident heterochely (Fig. 5D). M2 males have larger and wider chelipeds than M1 males, with a greater number of spines on the articles containing a more obtuse angulation. There is no gap between the fixed finger and the movable finger, and it is possible to observe a scarce pubescence in the palm of the propodus. Heterochely is more pronounced than in M1 (Fig. 5F). M3 males have notably more robust chelipeds, with the propodus, carpus, and merus having a rounded shape, especially in the propodus. Robust spines are present along the entire cheliped with an approximately orthogonal angulation, mainly in the propodus. There is an evident formation of a gap between the fixed finger and the movable finger, in addition to the presence of tufts of setae on the entire Table 2. Macrobrachium olfersii (Wiegmann, 1836). Results of analysis of covariance (ANCOVA) of logarithmized morphometric variables Relation Groups Parameters (log) F p IL vs. CL J vs. M1 a21.23 < 0.05 b2.21 0.13 M1 vs. M2 a3.96 < 0.05 b3.14 0.07 M2 vs. M3 a18.25 < 0.05 b0.38 0.53 ML vs. CL J vs. M1 a67.06 < 0.05 b0.14 0.70 M1 vs. M2 a83.55 < 0.05 b1.75 0.18 M2 vs. M3 a242.60 < 0.05 b2.69 0.10 CaL vs. CL J vs. M1 a47.83 < 0.05 b0.05 0.81 M1 vs. M2 a83.31 < 0.05 b3.53 0.06 M2 vs. M3 a212.62 < 0.05 b1.87 0.17 PrL vs. CL J vs. M1 a113.51 < 0.05 b3.14 0.07 M1 vs. M2 a89.31 < 0.05 b0.74 0.38 M2 vs. M3 a121.86 < 0.05 b0.66 0.41 DL vs. CL J vs. M1 a84.50 < 0.05 b0.92 0.33 M1 vs. M2 a95.94 < 0.05 b0.92 0.33 M2 vs. M3 a98.88 < 0.05 b0.29 0.58 PrH vs. CL J vs. M1 a  b0.30 < 0.05 M1 vs. M2 a102.44 < 0.05 b1.05 0.30 M2 vs. M3 a248.54 < 0.05 b0.001 0.97 ChL vs. CL J vs. M1 a96.40 < 0.01 b0.24 0.62 M1 vs. M2 a104.51 < 0.05 b2.25 0.13 M2 vs. M3 a206.69 < 0.05 b1.01 0.31 CL = carapace length; IL = ischium length; ML = merus length; CaL = carpus length; PrL = propodus length; DL = dactylus length; PrH = propodus height; ChL = major cheliped length. page 8 of 18Zoological Studies 61:83 (2022) © 2022 Academia Sinica, Taiwan inner surface of the fingers. The palm region of the propodus presents a thick layer of pubescence (Fig. 5H). Heterochely in M3 is the most evident among morphotypes of M. olfersii. Kruskal-Wallis analysis showed that there are differences in the angulation of cheliped spines among morphotypes (M1, M2, and M3) (H = 45.82; p < 0.01), with all morphotypes differing from each other (Dunn, p < 0.01) (Table 5). Geometric morphometrics A total of 194 shrimps were analyzed in the geometric morphometric analysis: 66 individuals from M1, 95 individuals from M2, and 33 individuals from M3. Propodus shape variation Statistical differences in propodus shape were observed among all male morphotypes (MANOVA; Wilk’s lambda = 0.2345; F = 15.98; p < 0.001). The analysis accurately separated M1, M2, and M3 individuals with efficacy of 80%, 73%, and 93%, respectively. The CVA also showed differences in the propodus shape between morphotypes, with an overlap between morphotypes 1 and 2 higher than the morphotypes 2 and Fig. 4. 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