A total count of 319 fish on Long Key was dominated by H. unicolor (63.9% of total) and H. gemma (22.3%), with H. puella (6.9%) and H. nigricans (5.3%) being less common, and tan hamlets (0.9%) and an undescribed olive green color morph (0.6%) being rare. In our smaller survey off Key Largo (n= 90 fish), H. puella was more common (17%) and H. nigricans was absent, whereas H. unicolor and H. gemma dominated both populations at roughly the same frequencies.
Hypoplectrus gemma and H. unicolor were found together on 36 of 39 transects. The relative frequencies of H. gemma and H. unicolor were random across the 39 transects (G= 44.075, df = 38, P > 0.1), indicating that morphospecies were not spatially isolated on different reefs in the daytime.
In 14 of 15 like pairs observed for the entire duration of their association, at least one spawn followed, and 13 of those pairs engaged in multiple spawns. The single unlike pair was likewise observed to spawn five times before separating. Hence pairing served as a good surrogate for spawning, as earlier more detailed behavioral observations also confirmed (Fischer 1980b). A total of 66 mating pairs were observed, and only one of these was a mixed pair (Table 1). The number of like pairs was far higher and the number of unlike pairs was far lower than expected under random pairing (G= 140.1, df = 6, P < 0.001, Table 1). Highly positive assortative mating was also evident in matings of uncommon morphospecies. Only three tan hamlets were seen on daytime transects, yet two were observed as a pair that spawned six times. Most notably, we saw two olive-colored fish not fitting any previously described morphospecies several hundred meters apart during the daytime; in the evening, a pair of these fish was seen spawning.
Table 1. Assortative pairing between Hypoplectrus morphospecies off Long Key, Florida. Expected numbers of pairs are calculated from binomial encounter frequencies based on relative abundances on Long Key reefs (see Results). A G-test for good ness-of-fit, with all categories of mixed pairs pooled for analysis, confirmed that mating was highly nonrandom (G=140.1, df=6, P<0.001).
| H. gemma×H. gemma||17||3.260|
| H. unicolor×H. unicolor||39||26.90|
| H. puella×H. puella|| 2||0.314|
| H. nigricans×H. nigricans|| 5||0.187|
| Tan×tan|| 1||0.006|
| Unknown×unknown|| 1||0.003|
| H. gemma×H. unicolor|| 0||18.80|
| H. gemma×H. puella|| 1||2.030|
| H. gemma×H. nigricans|| 0||1.570|
| H. gemma×tan|| 0||0.276|
| H. gemma×unknown|| 0||0.184|
| H. unicolor×H. puella|| 0||5.820|
| H. unicolor×H. nigricans|| 0||4.500|
| H. unicolor×tan|| 0||0.794|
| H. unicolor×unknown|| 0||0.529|
| H. puella×H. nigricans|| 0||0.485|
| H. puella×tan|| 0||0.086|
| H. puella×unknown|| 0||0.057|
| H. nigricans×tan|| 0||0.066|
| H. nigricans×unknown|| 0||0.044|
| Tan×unknown|| 0||0.008|
In the two individuals for which AFLP assays were replicated, 99.1% of 445 bands were reproducible. From 10 AFLP primer pairs across 12 individuals, a total of 949 distinct fragments were generated, of which 692 (72.9%) were polymorphic (i.e., present in at least one but not all individuals). Despite high polymorphism, no fragments were diagnostic, and NJ analysis (not shown) revealed no tendency for individuals to cluster according to morphospecies. Mean (± standard error) pairwise genetic distance was 0.046 (± 0.002) within H. gemma, 0.038 (± 0.001) within H. unicolor, and 0.044 (± 0.001) between morphospecies.
When 35 individuals were assayed across the two potentially most discriminating primer pairs, 288 fragments were generated, 253 (87.8%) of which were polymorphic. Of these, five fragments showed significant frequency differences between morphospecies (Table 2), but only one remained significant after sequential Dunn–Sidák correction for multiple testing (adjusted P= 0.000203 after 253 tests, Sokal and Rohlf 1995). This fragment was present in 12 of 18 H. unicolor, but absent in all 17 H. gemma. Consistent with results from exact tests, the θ estimator of FST, calculated using TFPGA and based on Lynch and Milligan's (1994) Taylor expansion estimate of allele frequencies, indicated moderate differentiation between morphospecies (θ= 0.0524, 95% confidence limits = 0.0318, 0.0739). The distribution of θ was “L-shaped,” with θ≤ 0 across 139 loci, 0 ≤θ≤ 0.1 across 89 loci, 0.1 ≤θ≤ 0.3 across 17 loci, and 0.3 ≤θ≤ 0.5 across only eight loci.
Table 2. AFLP fragments with significant frequency differences between Hypoplectrus gemma and H. unicolor. Values in the third and fourth columns are the number of individuals in which the fragment was present divided by the number of individuals assayed.
|Primer pair||Fragment size (bp)||H. gemma||H. unicolor|
NJ analysis (Fig. 1) revealed no clusters that were concordant with morphospecies boundaries. Individuals sorted into two clusters. Cluster 1 contained 29 of 35 fish (14 H. gemma, 15 H. unicolor) and no morphospecies-specific subclusters. Fourteen AFLPs distinguished cluster 1 from cluster 2 (bootstrap support = 99%), which contained three members of each morphospecies.
Figure 1. Unrooted NJ phylogram of Hypoplectrus gemma and H. unicolor AFLPs. Branch lengths are Nei and Li (1979) genetic distances estimated from 288 AFLP fragments generated using two selective primer combinations. Values label nodes with bootstrap support (1000 replicates) > 70%.
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In contrast to phenetic analysis, assignment tests correctly assigned most individuals to groups of like color pattern. Based on likelihood scores, 13 of 17 H. gemma and 13 of 18 H. unicolor were correctly assigned, whereas two H. gemma and three H. unicolor were incorrectly assigned, and two H. gemma and two H. unicolor were below the threshold Δ log L of 1 (Fig. 2). P-values from the allocation procedure (Fig. 2) sorted individuals into three classes. Individuals in the first class showed significant (P > 0.05) allocation only to their morphospecies of origin, whereas individuals in the second class received significant allocation to both morphospecies. This second outcome, suggesting some ambiguity in assignment, occurred with 12 of 17 H. gemma, and eight of these fish showed “high” (P > 0.5) heterospecific allocation values. In contrast, fewer (8 of 18) H. unicolor showed significant heterospecific allocation, and only one fish showed high heterospecific P-values. Along with greater Δ log L for H. unicolor, this pattern seems to reflect greater distinctiveness of H. unicolor fingerprints.
Figure 2. Assignment of individuals to morphospecies based on AFLP fingerprints. The top panels show the probability (P) of allocation and the bottom panels show log-likelihood differences favoring assignment to either morphospecies. Asterisks indicate individuals with relatively divergent AFLP fingerprints (see Fig. 1) and for which P-values for allocation to both morphospecies were low.
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The third class of individuals, each of which belonged to phenetic cluster 2 noted above (Fig. 1), showed low P-values (< 0.05) for allocation to both morphospecies. Although all but one of these six individuals were correctly assigned based on Δ log L, low P-values and low absolute values of log L for both morphospecies suggest that the fingerprints of these fish (see asterisks in Fig. 2) did not conform well to either morphospecies. The absence of significant allocation to either group suggests that these may be migrants from other, genetically distinctive subpopulations. To determine whether poorer allocation of H. gemma, noted above, was caused by the presence of these atypical fish, we reran the AFLPOP analysis with them removed. Five of the 14 remaining H. gemma but none of 15 H. unicolor received significant allocation to the heterospecific group. Hence, the greater genetic distinctiveness of H. unicolor remains, and cannot be attributed solely to data heterogeneity due to the atypical fish. Finally, the AFLP protocol was repeated with new DNA extracts from the six individuals from cluster 2. All fragments were reproduced, so we can exclude PCR artifacts as explanations for the uniqueness of these six fish.