Trophic differentiation in the phylogenetically young Cyprinodon species flock (Cyprinodontidae, Teleostei) from Laguna Chichancanab (Mexico)

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1 Biological Journal of the Linnean Society, 25, 85, With 4 figures Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society24-466The Linnean Society of London, 25? 25 85? Original Article TROPHIC DIFFERENTIATION IN A CYPRINODON SPECIES FLOCK J. HORSTKOTTE and U. STRECKER Trophic differentiation in the phylogenetically young Cyprinodon species flock (Cyprinodontidae, Teleostei) from Laguna Chichancanab (Mexico) JOACHIM HORSTKOTTE and ULRIKE STRECKER* Zoological Institute and Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 2146 Hamburg, Germany Received 24 February 24; accepted for publication 1 August 24 Analysis of the gut contents of six syntopic Cyprinodon species from Laguna Chichancanab, Mexico, shows that the supposed basal form, C. beltrani, feeds on detritus and has the longest gut. All other species have significantly shorter guts. Whereas C. simus exclusively ingests detritus despite short gut length, all other species additionally rely on different benthic invertebrates. Each species has developed specific preferences: C. labiosus (amphipods), C. verecundus (bivalves), C. maya (ostracods and gastropods) and C. esconditus (ostracods and chironomid larvae). However, all of them ingest a minimum of c. 4% detritus. According to Schoener s index, pairwise niche overlap varies from c. 4% to over 95% in the species flock. Contrary to this, there is almost no overlap between the members of the flock and the only other native fish species, Gambusia sexradiata, which feeds nearly exclusively on terrestrial arthropods dropping on the water surface. Recently, the cichlid genus Oreochromis and the characid Astyanax fasciatus gained access to the lake. The cichlids show moderate to high niche overlap, whereas in A. fasciatus it is low.. ADDITIONAL KEYWORDS: adaptive radiation diet - niche overlap pupfish - Schoener s index sympatric speciation. INTRODUCTION The evolutionary development of fish species flocks provides a unique opportunity to study mechanisms of adaptive radiation and speciation. A main factor leading to this development seems to be the existence of a depauperate fauna with open niches within the lake system prior to the adaptive radiation (Rosenzweig, 1978; Kondrashov & Mina, 1986; Wilkens, 1988; Doebeli, 1996; Strecker et al., 1996). Depauperate faunas may result from extreme abiotic conditions, such as, for example, low water temperatures, observed in Northern Eurasian temperate lakes (Arctic charr, Salvelinus alpinus (L.) in Lake Thingvallavatn, Iceland: Jónasson, 1992, 1993, 1998), northern American lakes (whitefish genus Coregonus: Schluter, 1996; Turgeon, Estoup & Bernatchez, 1999) or in cool, highaltitude tropical lakes (Orestias in Lake Titicaca, *Corresponding author. strecker@zoologie.uni-hamburg.de Peru: Kosswig & Villwock, 1964; Parenti, 1984; Villwock, 1986; Dejoux, 1994). A limited spectrum of species available to colonize new habitats may be another cause of the development of species flocks (Wilkens, 1988). This is exemplified by the cichlids of the Great East African lakes (Lowe-McConnell, 1993) or the species of the genus Puntius of Lake Lanao in the Philippines (Wahl, 1972; Kornfield & Carpenter, 1984). In Laguna Chichancanab, Yucatan Peninsula, Mexico, elevated salinity provides environmental conditions that cannot be tolerated by most freshwater species (Strecker, 1996, unpubl. data). Correspondingly, submerged vascular plants are absent and there is a strongly reduced number of invertebrate species. The fish fauna comprises an endemic species flock of seven species belonging to the genus Cyprinodon (Cyprinodontidae) and only one other fish, Gambusia sexradiata (Poeciliidae) (Humphries & Miller, 1981; Humphries, 1984; Strecker, 22, in press). Two additional fishes have recently become established in the lake. African cichlids of the genus Oreochromis were 125

2 126 J. HORSTKOTTE and U. STRECKER introduced when they escaped from aquacultural facilities after Hurricane Gilbert in 1988, and the widespread characid Astyanax fasciatus invaded the lake in 1996 (Schmitter-Soto & Caro, 1997; Strecker, 22, unpubl. data). Molecular studies indicate that the species flock originated about 8 years BP (Strecker et al., 1996). This age is supported by geological data that suggest that this date coincides with a desiccation phase of the lake (Covich & Stuiver, 1974; Hodell, Curtis & Brenner, 1995). The flock is composed of seven species, C. beltrani, C. labiosus, C. maya, C. simus, C. verecundus, the recently described C. esconditus and C. sp. nov. A (Strecker, 22, in press) and a group not assignable to any of the described species and referred to as hybrids by Humphries & Miller (1981). Analysis of the diet of four species revealed great overlap between C. beltrani, C. labiosus and C. simus, with only C. maya showing little similarity to the other species (Stevenson, 1992). In this paper we present a study of trophic differentiation of six species and the hybrids. A gut content analysis was performed to determine level of diet overlap. We included the other fish species, G. sexradiata, A. fasciatus and Oreochromis, in order to analyse their diet overlap with the species flock. MATERIAL AND METHODS The fish were caught in Laguna Chichancanab in February 1975, July 1991, December 1992, December 1993, January 1996, April 1997 and March 1999 at five different sampling sites A, B, C, E and F (see Fig. 1 and Strecker, unpubl. data). In 1992, at sampling site C, one additional catch was performed north of the regular sampling site (C1). Cyprinodon beltrani: 1975: E (N = 2); 1991, C (N = 1); 1992, B (N = 12), C1 (N = 7), E (N = 19); 1993, E (N = 1); 1996, C (N = 12), F (N = 9); 1999, E (N = 4). C. simus: 1996, C (N = 2), F (N = 11); 1997, F (N = 8); 1998, C (N = 2), F (N = 2). C. labiosus: 1991, C (N = 1); 1992, B (N = 6), C1 (N = 1), C (N = 9), E (N = 4); 1993, E (N = 5); 1999, E (N = 12). C. verecundus: 1991, C (N = 7); 1992, B (N = 3), C1 (N = 5), C (N = 4), E (N = 23); 1997, B (N = 7), C (N = 7); 1999, E (N = 12). C. maya: 1992, B (N = 1); 1993, A (N = 4), B (N = 9); 1996, F (N = 9); 1997, B (N = 8); 1999, E (N = 12). C. esconditus: 1992, E (N = 5); 1993, E (N = 9); 1999, C (N = 7), E (N = 11). Hybrids : 1992, C1 (N = 1), E (N = 1); 1996, C (N = 12); 1999, E (N = 11). Gambusia sexradiata: 1991, D (N = 12); 1992, B (N = 14), C (N = 3), E (N = 7). Astyanax fasciatus: 2, G (N = 2). Figure 1. Map of Laguna Chichancanab, Mexico showing sampling sites. Oreochromis: 1997, D (N = 7). Sampling was by net (mesh width.5.5 cm) of c. 5 m length pulled by two people from about 5 m offshore to the shore. Only the first catch at each locality was used for gut content analysis. Maximum sampling depth was 1.2 m. The sister species C. artifrons was collected in Laguna Yucalpeten, Chuburna, near Progreso, Yucatan in March 1998 for gut length comparison. All specimens were fixated in 96% ethanol, preserved in 7% ethanol and deposited in the collection of the Zoological Museum of the University of Hamburg (ZMH). For gut content analysis the following data were obtained from each specimen: 1. standard length (SL) to the nearest mm; 2. gut length (GL) to the nearest mm; 3. number of prey specimens (if countable prey); and 4. dry weight (DW) of each dietary item (i) to the nearest.1 mg.

3 TROPHIC DIFFERENTIATION IN A CYPRINODON SPECIES FLOCK 127 We used dry weight to avoid errors caused by adherent water. Drying was done at 7 C overnight. To take into account differences in gut fullness we calculated the gut fullness index (GFI) as the ratio of the sum of dry weight of all food items (SD) and gut length (GL) for each specimen (x). GFI x SDx[ mg] = GL [ mm] Per cent dry weight of each item (PD i ) is calculated as a percentage of SD (Ross, 1977). PD i of each investigated fish is multiplied with GFI resulting in Relative dry weight (RD) of item i : SDx[ mg] DWi mg RDi = PD i[ %] GFIx = 1 GLx[ mm] [ ] SDx[ mg] DWi[ mg] = GL [ mm] 1 x RD shows how many mg of item i are associated with 1 mm of gut length. The index of relative importance IRI (Pinkas, Oliphant & Iverson, 1971; Prince, 1975; IRI = (N i [%] + V i [%]) F i [%], where N = number, V = volume and F = frequency of occurrence of item i, reviewed by Hyslop, 198), was modified to take into account the existence of countable and uncountable food items in the diet examined: DW IRI = [%] occurrence[ %] GL To measure niche overlap we used Schoener s index (Schoener, 197; Hurlbert, 1978; Wallace, 1981). The detritus was baked at 55 C to remove organic material from lime or gypsum sediments. Additionally, samples were placed under a scanning electron microscope to further analyse its composition. Statistical analysis was performed using SPSS v We performed an ANOVA followed by post hoc Scheffé tests for comparisons between the different species. RESULTS GUT LENGTH AND SPECTRUM OF FOOD ITEMS The mean relative gut lengths of C. maya, C. simus, C. labiosus, C. verecundus, C. esconditus and that of the hybrids ranged between 2. and 2.9, about half that of C. beltrani and C. artifrons (P.1) (Fig. 2). Mean relative gut length is also significantly differnt between C. simus and C. verecundus/c. maya as well as between C. esconditus and C. maya (P.25). The gut content was divided into the following components (Figs 3, 4; Table 1). Detritus Detritus occurred with a frequency of 1% in all species within the flock, except in C. verecundus, where it x Gut length/standard length C. artifrons (32) C. beltrani (98) C. simus (65) C. labiosus (81) C.verecundus (89) C. maya (63) C. esconditus (52) Hybrids (55) Figure 2. Relative gut length in Cyprinodon artifrons and the Cyprinodon species flock. Numbers of specimens in parentheses. was slightly lower (97.1%). It comprised almost the entire gut contents of C. beltrani and C. simus, as indicated by the high MRD and IRI values. The hybrids and C. esconditus also show relatively high values. In C. labiosus they are intermediate, while in C. verecundus and in C. maya they are the lowest (MRD 34. and 26.8, IRI 41.3 and 39., respectively). Detritus consisted of an inorganic and an organic component. The organic component included diatoms, bacteria, fungal hyphae and faecal pellets of bivalves (Lucina). The inorganic component was 4 45% in C. maya, C. beltrani and C. simus, and 33% in the remaining species. Amphipoda The benthic species Hyalella azteca, the only amphipod in Laguna Chichancanab (henceforth the lake), is frequently preyed upon by C. labiosus and C. verecundus (F 76.8 and 7.6, respectively). It was less important for C. beltrani, C. esconditus, C. maya and the hybrids (F , IRI..5) and was absent in C. simus. It is the most important prey item for C. labiosus (MRD 27.2 vs in C. verecundus; P.1). The specimens ingested have a body size of 3 mm. Ostracoda Seven species of ostracods occur in the lake. They dwell on the lake bottom or between the thalli of Chara (Strecker, 1996). They are the preferred animal prey of C. maya (MRD 19.3, F 86.4). It differs significantly from all other members of the flock (P.1 to.1). The only exception is C. esconditus (P =.17) for which it is one of the two most important animal items (MRD 1., F 87.5). Despite a frequency of occurrence of 5.6 and 36. in C. beltrani and

4 128 J. HORSTKOTTE and U. STRECKER Cyprinodon beltrani N = 85 Detritus Amphipoda Ostracoda Cyprinodon simus N = 25 Cyprinodonlabiosus N = 56 Hybrids N = 19 Cyprinodon maya N = 52 Figure 3. Prey items of members of the Cyprinodon species flock and of Gambusia sexradiata. Thickness of arrows corresponds to the mean relative dry weight, values below 1% not shown. C. simus the low IRI values (.5 resp..2) indicate that ostracods are relatively unimportant for these species, as well as for C. labiosus, C. verecundus and the hybrids (IRI ). The maximum body size of ingested specimens is 2 mm. Insect larvae Aquatic insect larvae (ephemeropterans, trichopterans and chironomids) constitute a considerable part of the prey of C. esconditus and C. verecundus (MRD 1.2% and 1.4%, F 84.4 and 86.8, respectively) differing significantly from all other Cyprinodon species (P.1.48), but not between each other and the hybrids (P = ). This food item is also frequently eaten by C. labiosus and the hybrids, but in smaller quantities (F 76.8 and 76., MRD 4.4 and 3.8, respectively). Insect larvae consumed by C. esconditus were mainly chironomids, whereas C. verecundus preferred ephemeropterans. For C. beltrani, C. simus and C. maya (IRI.2.8) larvae are not an important item, despite the moderate frequency in C. maya (F 5.). Decapoda and Acari Other aquatic arthropods like acarids (oribatei and hydrachnellae) were not found in C. simus and rarely Insect larvae (aquatic) Chara Gastropoda Bivalvia Teleostei Arthropoda (terrestrial) Cyprinodon esconditus N = 25 Cyprinodon verecundus N = 68 Gambusia sexradiata N = 36 in all other species (F , IRI.1). Parts of the decapod crustacean Procambarus llamasii were found in C. maya in relatively low frequency (F 13.5), but this is a minor food item (IRI.1). Arthropoda (terr.) Terrestrial arthropods (adult ants, dipterans, coleopterans, zygopterans and spiders), were absent in C. esconditus and the hybrids and rarely occurred in the other species (F , IRI.3). Gastropoda The snail Pyrgophorus coronatus (Hydrobiidae) was most common in C. maya (F 63.5, IRI 15.8), a significant difference to all other Cyprinodon species (P.1.37). It is consumed to a moderate degree by C. labiosus and C. verecundus (F 48.2 and 54.4, respectively), but was essentially absent in the other species (IRI..2). A larger gastropod (up to 8 mm body size), the rare Physella cubensis, was eaten only by C. maya. Bivalvia Lucina sp. (Luciniidae), the only bivalve mollusc in the lake, occurs between the basal leaves of the sedges or deep in Chara pads. It is a significant diet

5 TROPHIC DIFFERENTIATION IN A CYPRINODON SPECIES FLOCK 129 Table 1. Means of relative dry weight (MRD), frequency of occurrence (F) and index of relative importance (IRI) (Pinkas et al., 1971; Hyslop, 198, modified) of food items in the Cyprinodon species flock, Gambusia sexradiata, Astyanax fasciatus and Oreochromis. Number of individuals studied are in parentheses * terrestrial C. beltrani C. simus C. labiosus C. verecundus C. maya C. esconditus hybrids G. sexradiata A. fasciatus Oreochromis (85) (25) (56) (68) (52) (32) (25) (36) (2) (7) MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI MRD F IRI Detritus Amphipoda < Ostracoda Insect larvae Decapoda Acari < < < Arthropoda* Gastropoda < Bivalvia < Bryozoa Teleostei < Lyngbia < < Chara Dry weight [%] Dry weight [%] Dry weight [%] Dry weight [%] Dry weight [%] Dry weight [%] Dry weight [%] Cyprinodon beltrani N = 85 Cyprinodon simus N = 25 Cyprinodon labiosus N = 56 Cyprinodon verecundus N = 68 Cyprinodon maya N = 52 Cyprinodon esconditus N = 32 Hybrids N = 25 Figure 4. Percentage of dry weight of food items in the Cyprinodon species flock. 1. Detritus. 2. Amphipoda. 3. Ostracoda. 4. Insect larvae (aquatic). 5. Arthropoda (terrestrial). 6. Gastropoda. 7. Bivalvia. 8. Teleostei. 9. Chara. item for C. verecundus (F 72.1, IRI 32.1, P.1), which preys on 2 3 mm juveniles. Considerable amounts are also ingested by C. maya (F 23.1, IRI 5.9), which is able to ingest larger specimens up to 6 mm in diameter. Bivalves are unimportant in the diets of the other species. The relatively high MRD value (12.5) for bivalves in hybrids is due to a single individual having consumed an unusually large number (82.4% of the DW) (Fig. 4) which is reflected by a low IRI (1.8). Bryozoa Bryozoans (Plumatella) were eaten only by A. fasciatus. Teleostei Fish prey, mainly G. sexradiata, is a negligible food item for most species of the flock. It was not found in C. simus, C. labiosus, C. esconditus, or the hybrids. Two and five juvenile Gambusia, 7 mm in total length,

6 13 J. HORSTKOTTE and U. STRECKER were found in C. beltrani and C. verecundus, respectively (IRI.; F 2.4, 2.9). Fish prey was more important in C. maya (F 28.8, IRI 3.4), which had ingested Gambusia and Cyprinodon (two were C. simus) juveniles. The largest fish prey was 26 mm SL. Lyngbia and Chara Algae consumed were primarily Chara and Lyngbia. Parts of Chara thalli were most abundant in C. maya (F 86.5, IRI 1.), moderately common in C. beltrani (F 44.7, IRI 1.6), and unimportant in the other species and the hybrids (F , IRI.7). Lyngbia was absent in C. simus and occurred only in trace amounts in C. beltrani, C. labiosus, C. verecundus, C. esconditus and the hybrids (IRI ). It was most abundant in C. maya (F 23.1, IRI.4). FOOD SPECTRUM OF G. SEXRADIATA AND RECENTLY INTRODUCED FISH (A. FASCIATUS AND CICHLIDS) G. sexradiata feeds almost exclusively on terrestrial arthropods accidentally dropping onto the water surface (F 97.2, IRI 99.) (Fig. 3; Table 1), although it also consumes small amounts of ostracods, gastropods and teleosts (IRI.1.7). A. fasciatus has, in comparison, a broader food spectrum (Table 1). The primary items were terrestrial arthropods and aquatic insect larvae (F 6. and 7., IRI 59. and 24.). Ostracods, bryozoans and detritus occur at moderate frequency (F 3. 5.) and are of minor importance (IRI 7.2, 5. and 1.8, respectively), as were items like amphipods, decapods, algae (Chara), teleosts and acarids (F , IRI. 1.4). Two items, bryozoans (Plumatella) and oligochaetes, were eaten only by Astyanax. The introduced cichlids are detritivorous, with gut lengths sevenfold greater than SL. Their main food item was detritus (F 1, IRI 99.8), the organic component of which maximally accounted for 4% of the total mass of sediment. The average grain size of the inorganic component was about double the size of that of the species flock. There were also small amounts of ostracods, terrestrial arthropods and algae (Chara) (IRI..1) (Table 1). DIET OVERLAP The highest diet overlap in the flock occurred between C. beltrani and C. simus (98%, Table 2), closely followed by C. beltrani, C. simus, C. esconditus and the hybrids (82 91%). Moderate overlap occurred among C. labiosus and C. beltrani, C. simus, C. esconditus, and hybrids (66 72%). In general, comparisons involving C. maya and C. verecundus were lowest, with the least overlap occurring in comparison with C. simus and C. beltrani (4 43%). Table 2. Diet overlap (Schoener, 197) in the Cyprinodon species flock, Gambusia sexradiata, Astyanax fasciatus and Oreochromis from Laguna Chichancanab. Number of individuals examined are in parentheses C. beltrani C. simus C. labiosus C. verecundus C. maya C. esconditus Hybrids G. sexradiata A. fasciatus C. beltrani (85) C. simus (25).985 C. labiosus (56).661 C. verecundus (68) C. maya (52) C. esconditus (32) Hybrids (25) G. sexradiata (36) A. fasciatus (2) Oreochromis (7)

7 TROPHIC DIFFERENTIATION IN A CYPRINODON SPECIES FLOCK 131 Almost no diet overlap occurred between members of the species flock and G. sexradiata (.4 1.2%). Among the newly introduced species, A. fasciatus showed low overlap with the species flock ( %), while the cichlids had moderate to high overlap with the flock ( %) (Table 2). The cichlids showed little overlap with G. sexradiata and A. fasciatus (.2 and 1.9%, respectively), but overlap between the two latter species was markedly higher (58.7%). DISCUSSION FOOD NICHES The outstanding characteristic of the Cyprinodon species flock in Laguna Chichancanab is that detritus plays an essential role in the diet of all its members. Nonetheless, various members have turned toward greater carnivory, as indicated by their gut contents and shorter guts (Figs 2, 3). Contrary to expectation, however, C. simus has a short gut, yet we found only detritus in its diet. Earlier, Humphries & Miller (1981) observed C. simus feeding on plankters. The earlier studies have revealed that the lake now supports almost no zooplankton and only small amounts of phytoplankton (Elias-Gutierrez et al., 21; Strecker, unpubl. data). This change in food availability might explain the extreme population decline or extinction of C. simus in the lake (Strecker, unpubl. data). Perhaps it was not able to switch to an alternative animal prey item after as hypothesised before the zooplankton had disappeared. Therefore, it is possible that C. simus, as one of the carnivorous species of the flock, is in an evolutionary adaptational stage that no longer allows it to exist on detritus exclusively. In principle, every species of the flock, including the basal C. beltrani, may feed on all items. However, there appear to be specific preferences for different prey: amphipods for C. labiosus, ostracods and gastropods for C. maya, bivalves for C. verecundus and ostracods and aquatic insect larvae for C. esconditus. A similar kind of diet segregation occurs between two morphs of Percichthys trucha (Percichthyidae) in the Andes (Argentina). Ruzzante et al. (1998) reported that both fed primarily on benthic invertebrates, but the littoral morph fed more on larval anisopterans than did the deep benthic morph. Diet segregation was also found in three-spined sticklebacks, Gasterosteus sp. (Schluter, 1996). Similar observations were made for birds between two song groups of Darwin s finches, Geospiza conirostris (Grant & Grant, 1989). Almost all potential food resources of animal prey occurring in the lake are exploited by one of the carnivorous Cyprinodon species. There are some exceptions, though. The surface feeding niche has not been exploited, possibly because it was already occupied by G. sexradiata, supporting the hypothesis that open niches are the main cause for adaptive radiation (Wilkens, 1988; for a review see Schluter, 22). However, it is most remarkable that, within a lake with abundant masses of small fish, no specialized piscivore has yet developed. In contrast to this, a piscivorous bulldog form is found in another species flock of Cyprinodon on San Salvador Island, Bahamas (Holtmeier, 21). It is still an open question how species-specific preferences for a particular food item could have evolved (e.g. ostracods and gastropods in C. maya). It is possible that this is due to a focus on a specific prey item. Our study shows that all members of the flock may potentially feed on nearly every prey item occurring in the lake. We favour the hypothesis that specific habitat preferences may be responsible. For example, the main feeding habitat of C. maya is probably Chara vegetation, in which it finds ostracods and gastropods living on the thalli. This suggests that divergent behavioural characteristics have evolved, enabling the different species to track their preferential prey in its typical habitat. Thus, in the Cyprinodon flock, niche partitioning may not have been driven by searching for a specific food item but by searching for specific prey habitats. Major differences to a previous study (Stevenson, 1992) on diet composition of four of the species (C. beltrani, C. simus, C. labiosus and C. maya) include Stevenson s findings of high occurrence of Lyngbia (up to 71% in C. simus) as well as a relatively high occurrence of microscopic dinoflagellates and rotifers and a total lack of bivalves. Stevenson (1992) used Hynes s (195) point-method, which has been criticized because of its inaccuracy (see Hyslop, 198; Marrero & Lopez-Rojas, 1995; Nieder, 1997). The unusual occurrence of Lyngbia in Stevenson s (1992) study might be just a single event happening in the sampling year. The different pairwise combinations of species in the flock produce variations of between 4 and 99% in diet overlap. Less closely related fish species generally show much less overlap (Fujita et al., 1995; de Almeida, Hahn & de Vazzoler, 1997; Little et al., 1998), even when juvenile life stages are compared (Thiel et al., 1996). Also different morphs within another fish species flock (Salvelinus alpinus from Thingvallavatn, Iceland), exhibit overlaps below 47% [with the exception of one pair, a small and a large benthivorous morph, having higher values (66%): Sandlund et al., 1992; Snorrason et al., 1994; Skúlason & Smith, 1995]. The comparatively high diet overlaps in the species flock of Cyprinodon may reflect its youthful age, as indicated by mitochondrial DNA (mtdna) data (Strecker et al., 1996).

8 132 J. HORSTKOTTE and U. STRECKER The hybrids represent 45 6% of the flock (Strecker, unpubl. data). It is still unclear whether they are really hybrids, yet undetermined species and/ or just represent morphological plasticity (Strecker et al., 1996). This group has a short gut similar to the more carnivorous species (Fig. 2), yet, in contrast with those, there was no apparent preference for any particular item. This might be a result of this group being non-homogeneous in morphology and/or in the initial stage of specialization. The members of the species flock show different degrees of diet specialization (Table 2). C. verecundus and C. maya have the lowest niche overlap with the other members of the flock (42 61% and 4 51%, respectively). Overlaps smaller than 6% are considered significant resource partitioning (Zaret & Rand, 1971; Mathur, 1977; Wallace, 1981). The low overlap found in C. maya is congruent with reproductive isolation for this species, as concluded from mtdna data (Strecker et al., 1996) as well as behavioural studies (Strecker & Kodric-Brown, 1999, 2; Kodric-Brown & Strecker, 21). The other species cannot yet be discriminated genetically and show higher niche overlaps. However, the diet overlap of C. labiosus is close to the biologically significant value of 6%. This coincides with its intermediate level of reproductive isolation relative to C. beltrani and C. maya in mating experiments (Strecker & Kodric-Brown, 1999, 2; Kodric-Brown & Strecker, 21). Based on these results we assume that C. maya has reached the highest level of species segregation in the flock and that the differences described before might reflect varying evolutionary stages and/or that the species coexist and there may be no need for further specialization. INFLUENCES OF THE NEWLY INTRODUCED SPECIES ON THE SPECIES FLOCK The level of food overlap between A. fasciatus and the species flock is low (Table 2). The main impact of this predator is probably on the fry. This may help explain the reduced densities of Cyprinodon since its appearance in the lake (Strecker, unpubl. data). The lack of fish prey found in A. fasciatus may be due to the very efficient protein digestion provided by the stomachs of carnivores (Horn 1998) which, in contrast, is missing in Cyprinodon. The cichlids have high overlap with C. simus, C. beltrani, the hybrids and C. esconditus (Table 2). However, according to Pedersen (1999), potential competition for a food resource does not mean that it will always occur. Despite the envisaged probable extinction of C. simus since the introduction of the cichlids, C. beltrani still occurs at high frequency (Strecker, unpubl. data). Therefore, food competition for detritus between Cyprinodon and cichlids appears low. This is supported by the differences in grain size of the inorganic component of sediment material in the gut contents, indicating some kind of divergence in feeding. Whereas trophic competition from cichlids seems to play a minor role in the species flock, other factors such as increased parasitism and bioturbation seem to have greater impact (Strecker, unpubl. data). The decline or complete extinction of C. simus is not yet understood. As has been shown, food competition with the cichlids and A. fasciatus provides no probable cause. Although Humphries & Miller (1981) observed C. simus feeding on plankters, gut analysis of specimens from before the appearance of the introduced species revealed no plankters in the diet (Stevenson, 1992). Nonetheless, if C. simus is characterized as a planktivore, it cannot be ruled out that the decrease of one of its preferred foods (i.e. plankters) is the main cause for its decline. In other lakes the introduction of exotic fish species may have been responsible for dramatic changes to the local ecosystems. In several instances this has led to the extinction, in whole or in part, of fish species flocks (Kornfield & Carpenter, 1984; Ogutu-Ohwayo, 199, 1993; Goldschmidt, Witte & Wanink, 1993; Lowe-McConnell, 1993; Seehausen, van Alphen & Witte, 1997). It appears that in Laguna Chichancanab the members of the flock, except for C. simus, have survived, as indicated by a ten-year field study showing that following the introduction of the cichlids and A. fasciatus, they still occur at more or less stable relative frequencies (Strecker, unpubl. data). ACKNOWLEDGEMENTS We are grateful to Prof. Dr Horst Wilkens for valuable discussions. We thank Dr Steven J. B. Cooper, South Australian Museum, Adelaide for his assistance in improving the English and Hermann Müller for help with the statistics. 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