Morphological variability and distribution of the exotic Asian Mesocyclops thermocyclopoides ( Copepoda : Cyclopoida ) in the Neotropical region

From a series of biological samples collected from different freshwater environments in Costa Rica, Central America, the exotic Asian cyclopoid Mesocyclops thermocyclopoides Harada, 1931 was identified. We analyzed the morphology and appendage ornamentation of different Neotropical populations of this species, including specimens from Honduras, southeastern Mexico, and Costa Rica. We also examined Asian specimens from Taiwan, Indonesia, Vietnam, and Thailand, and performed a comparison of the Neotropical and Asian populations including a Principal Component Analysis (PCA). The Neotropical and Asian specimens show subtle morphological variations in the antennules, antennae, mandibles, swimming legs 1-4, and fifth legs. Some characters in the Neotropical group appear to diverge from the Asian pattern and the PCA indicated that intercontinental populations of M. thermocyclopoides are far from being homogeneous. These intra-specific differences are described to expand the known morphological range of this species and to provide the first comparative analysis of an exotic copepod in the Americas. Our analysis suggests that the geographic isolation of the American populations and the subtle morphological divergences with respect to the Asian patterns could be related to speciation processes in the Neotropical region, but also intra-Asian differences are reported. In the Neotropical region this species appears to be restricted to southeastern Mexico, Central America, and one Caribbean island; its potential as biological control of mosquito might enhance its spread in the region.

The specimens from Costa Rica were identified following the keys and illustrations by SUÁREZ-MORALES & GUTIÉRREZ-AGUIRRE (2001), GUTIÉRREZ-AGUIRRE et al. (2003), andHOLYNSKA et al. (2003).The morphological analysis of these specimens revealed some differences with respect to specimens from other Neotropical populations, and from the Asian pattern, as described below.
Morphological remarks and comparisons.The main differences between populations from Asia (cf.HOLYNSKA 1994, HOLYNSKA et al. 2003; museum specimens from different geographic areas in Asia), southeastern Mexico (two localities at the state of Tabasco, southeastern Mexico), Honduras (cf.GUTIÉRREZ-AGUIRRE et al. 2003; specimens from El Progreso), and Costa Rica (specimens from El Arenal) are presented in detail in Table I and summarized in Table II.The size of the body of individuals in these populations (excluding caudal rami) differ among the Neotropical populations examined: specimens from Costa Rica are the smallest (0.78-0.80 mm), those from Honduras are the largest (0.97-1.0 mm); specimens from two localities in southeastern Mexico have intermediate size ranges (Báscula: 0.74-0.89mm; Pulsar 0.90-1.0mm).Overall, the Asian specimens tend to be larger than the Neotropical forms; the smallest sizes were found in Vietnam II (0.73-0.88) and the largest were from Thailand (0.95-1.26 mm) and Indonesia (1.01-1.30mm).
Additional differences among the Neotropical populations include the ornamentation of the antennular segments 4 and 7, as follows: the specimens from Costa Rica and Asia bear four rows of spinules on the fourth antennular segment vs. two in both the Honduran and Mexican populations (Fig. 1).On the seventh antennular segment, the Asian specimen has six groups of tiny spinules, vs. five such groups in both Mexico and Honduras and nine in the Costa Rican specimens.
The ornamentation of the antennal basis includes several rows of spines which have been marked and denominated by their position following GUTIÉRREZ-AGUIRRE et al. (2003).The range of the number of spines per row is different in all groups of specimens examined (Tabs I and II).None of the Neotropical populations have less than seven and more than 11 spines in row h.Only the Asian populations examined have less than six spines in row i.We did not consider the number of spinules in row c because it often continues on the anterior surface, making counting difficult.We observed, however, that the specimens from Costa Rica (Fig. 2) have clearly larger and stronger spinules in this row when compared with the Asian, Mexican, and Honduran populations (GUTIÉRREZ-AGUIRRE et al. 2003, HOLYNSKA et al. 2003).The length ratio of the medial basipodal setae (arrowed in Fig. 2) is alike in the Neotropical populations (Mexico, Honduras, Costa Rica) (1:1) and differs from the Asian figure: 1.2-1.47:1(n = 12).
The mandible gnathobase and the teeth number show clear interspecific variation in Mesocyclops (SUÁREZ-MORALES et al. 2003).In line with that prediction, we found some differences among the Neotropical populations investigated .Specimens from Báscula (Tabasco, Mexico) have nine teeth vs. eight in the Costar Rican, Honduran and Pulsar (Tabasco, Mexico) populations (Figs 13-16); among the latter populations, only the Costar Rican specimens do not bear accessory spines.The mandibular size relative to the body size (MSI index, SUÁREZ-MORALES et al. 2003) was measured; the Costar Rican (3.17) and Báscula (3.11) populations showed the highest MSI values.Lower values were found in the Honduran specimens (2.79) and in the population from Pulsar (Tabasco) (2.67).These mandibular characters were not evaluated in the Asian specimens.tal end of the spine (arrow in Fig. 5).Also, the inner and outer endopodal setae of the maxillular palp are subequal in the Asian, Mexican, and Honduran populations, but the medial seta is clearly shorter than the other setae in specimens from Costa Rica (arrowed in Fig. 4).Ornamentation and elements of the maxilla are practically the same in all specimens (Fig. 6).
The coxal ornamentation of P1 is similar in all groups examined.The populations from Honduras, Mexico, and Asia all have a semi-circular row of spines on the anterior surface of the distal margin of the basipodite, near the insertion of the exopodite (arrowed in Fig. 7).Furthermore, specimens from Mexico and Honduras also have a row of tiny spinules near the insertion of the endopodite.In the Costa Rican specimens (Fig. 7), the inner coxal seta reaches the distal margin of the first endopodal segment, whereas this seta is distinctly longer in the Asian, Mexican, and Honduran populations, reaching the distal margin of the second endopodal segment (see HOLYNSKA et al. 2003).The length ratio of the apical endopodal seta/segment length is 0.71 in Asian specimens vs. 1.04 in Costa Rican specimens (Fig. 7).The inner apical seta of this segment also differs between populations; in Asian specimens this seta is clearly longer than the inner spine, being relatively shorter in the Costa Rican specimens.The seta on the lateral margin of the same segment is longer than the apical spine in Asian specimens, whereas this spine barely exceeds half of the length of the spine in specimens from Costa Rica (arrowed in Fig. 7).
The ornamentation of both the coxopodite and basipodite of the second swimming leg differs among the examined populations.Specimens from Costa Rica bear only a single row of hair-like setae on the outer coxal margin (Fig. 8), whereas specimens from Mexico and Honduras have three additional transverse rows of setal elements on the frontal surface (GUTIÉRREZ-AGUIRRE et al. 2003).The ornamentation of the third swimming leg also differs among the three Neotropical groups of specimens; all populations bear a row of hair-like setae on the outer coxal margin, but the Mexican and Hondu-ran specimens have a second row of shorter hair-like setae on the proximal surface of same inner margin (see GUTIÉRREZ-AGUIRRE et al. 2003: fig. 3b).Another difference is the position and size of a transverse row of hair-like setae along the distal margin of the coxopodite.In both the Mexican and Honduran specimens this row is located medially near the insertion of the coxopodite, whereas in the Costa Rican specimens the position of this row is on the middle coxal surface and closer to the outer margin (arrowed in Fig. 9).
The coxal ornamentation of the fourth swimming leg is almost the same in all populations examined (Fig. 10).The four groups of specimens have a group of hair-like setae on the inner margin of the basipodite, including a transverse row of hair-like setae close to the insertion of the inner coxal seta.Differences in P4 ornamentation and proportions include the ranges of length ratio of the apical spine/third endopodal segment, length/width ratio of the same segment, and inner spine/ outer spine ratio (Tabs I and II).Only in the Asian populations less than six spines on the basal margin of coxopodite have been observed.Additionally, specimens from Mexico and Honduras have a unique pattern of ornamentation of tiny spinules on the anterior surface of the three endopodal segments.This pattern was not observed in the Costa Rican or in the Asian populations.
The length ratio of the inner spine/outer seta of the fifth leg was also evaluated; all populations examined have variable ratios (Fig. 11, Tabs I and II).The proportion and relative lengths of the setal elements and the ornamentations of the caudal rami of specimens from Costa Rica, Mexico, Honduras and specimens from Asia are within the range known from the type Asian population (HOLYNSKA 1994, HOLYNSKA et al. 2003).
In order to define the extent and relevance of the differences found among the different populations of M. thermocyclopoides examined, we performed a Principal Component Analysis (PCA), which is a statistical tool commonly used to evaluate the morphometric and meristic characters among populations ).We used the PRIMER 6 software with log 10 -transformed meristic and morphometrical data.The resulting plot (Fig. 17) included all individuals of the different populations examined.The first principal component (PC1) accounted for 42.2% of the variation, the second (PC2) for 20% among the samples (Tab.III).The plot of the component variants PC1 and PC2 (Fig. 17) shows a discrimination of population scores of different groups along PC1, including the Asian populations from Indonesia (marked as "In" in Fig. 17) with highest scores and also the two Thailand populations (TA and TB) with lower ones.Also, the Mexican populations are aligned along the PC2, thus forming a uniform group.The Honduran specimens clearly diverge in character 2, number of spines on row "i" of the antennal basis.The other Central America specimens, from Costa Rica, appear to be more closely related to the Mexican pattern that to the Honduran or Asian ones.Among the Asian populations, there is a divergence between the Indonesia and Thailand forms and the Vietnam (Vi1, Vi2) and Taiwan (Tw) populations (Fig. 17).Examination of the distance of variables from the origin revealed that the main observed differences (characters 1 and 2) were related to the number of spines on the rows of the antennal basis.Characters 6 and 7, the ratios of length/width of the third endopodal segment of the fourth leg and the inner/outer spines of the same segment, respectively, were also important.Characters 8 and 9 (inner spine of the fifth leg and ratio of the outer apical spines of the third endopodal segment of the fourth leg) have no detectable variations.

DISCUSSION
Because of the rarity of cosmopolitan forms in Mesocyclops, earlier Neotropical records of M. thermocyclopoides were suspected to represent undescribed taxa, but GUTIÉRREZ-AGUIRRE et al. (2003) confirmed the presence of the strict form of this species in the Neotropics.Our survey provides detailed morphological information about subtle differences among Neotropical and Asian populations of this species.Intraspecific morphological variations among some cyclopoids have been related to seasonal   changes, but such variations in presumably cosmopolitan species are, in many species, related to the formation of species complexes (LEE 2000, MIRABDULLAYEV & DEFAYE 2003).
Overall, the morphological differences described herein are not consistently related to either the Neotropical or the Asian populations groups of M. thermocyclopoides.Our analysis showed that the characters evaluated, which are usually those used to distinguish species within the genus, show subtle but detectable intra-specific differences among the populations examined; it is clear that these meristic and morphometric characters are not uniform throughout the species range.There is some indication that the American populations are diverging from the Asian ones and that even among the Asian forms there is some degree of differentiation, particularly between the Indonesia-Thailand and Taiwan-Vietnam groups.Among the Neotropical groups, the Honduran diverges at least in one of the most variable characters, and the Mexican and Costa Rican specimens are more closely related to each other than to most of the other groups examined.Overall, the geographic isolation and the presumed reproductive isolation of the immigrant Neotropical populations, and also the subtle but consistent morphological divergence with respect to the Asian populations, suggest that a process of speciation could be occurring among the American populations of M. thermocyclopoides, particularly in Central America and southern Mexico.This isolation is expressed in subtle differences not only between the continents but also within them.
Originally described from Taiwan, the known distributional range of M. thermocyclopoides in Asia includes Japan, southern China, Burma, Vietnam, Thailand, Indochina, Malaysia, Indonesia, and Java (HOLYNSKA 1994, GUO 2000, HOLYNSKA et al. 2003).American records from Central America and the Caribbean most probably represent introduced populations (GUTIÉRREZ- AGUIRRE et al. 2003, HOLYNSKA et al. 2003).In Central America M. thermocyclopoides was first recorded from Costa Rica by COLLADO et al. (1984), from 14 localities, and later on by HERNÁNDEZ-CHAVARRÍA & SCHAPER (2000).It has also been recorded from Puerto Rico (MARTEN, 1994), Honduras (MARTEN et al. 1994 et al. 2011), thus confirming the growing occurrence of populations of exotic copepods in the neotropics (Fig. 18).
Other exotic Asian or Afro-Asian Mesocyclops known in the Neotropical region are M. aspericornis (SUÁREZ-MORALES et al. 2011) and M. pehpeiensis (SUÁREZ-MORALES et al. 2005, MENÉNDEZ-DÍAZ et al. 2006).These exotic species have been successfully tested as biological control agents of mosquito larvae.Mesocyclops thermocyclopoides has been deemed an efficient biological control agent of mosquito larvae in Asia (KUMAR & RAO 2003) and also in the Americas (SOTO et al. 1999); its life cycle has important advantages over that of congeners in terms of maturation (SUÁREZ-MORALES et al. 2007).Other native Neotropical Mesocyclops have ranked low as potential mosquito control (TRANCHIDA et al. 2009) agents.Despite the risks involved in the introduction and spread of biological controls (SIMBERLOFF & STILLING 1996), it is expected that the success and potential use of M. thermocyclopoides for the biological control of mosquitoes could favor its spread into other tropical regions of the Americas.(MARTEN et al.1994;GUTIÉRREZ-AGUIRRE et al. 2003); 3) different areas of Costa Rica (COLLADO et al. 1986); 4) El Arenal, Costa Rica (this work); 5) Puerto Rico (MARTEN 1994).

Table II .
Summarized data of the variation of appendage ornamentation and other morphological characters among three different groups of populations of Mesocyclops thermocylopoides.(P4) Fourth swimming leg, (End3P4) third endopodal segment of fourth swimming leg.

Table III .
Results of the PCA, including eigenvalues and percentages of variability explained for PC's of populations of Mesocyclops thermocyclopoides.