RESULTS

RESULTS

Phylogenetic analyses

The MRBAYES and BEAST produced similar topologies. Bayesian inference recovered almost all genera with posterior probabilities>0.95 ( Fig. 3 View Figure 3 ). Most genera were monophyletic except the following: (i) Xenotoca variata (Bean, 1887) was sister to Ameca splendens Miller & Fitzsimons, 1971 and placed well outside genus Xenotoca Hubbs & Turner, 1939 ; (ii) Jordanella pulchra (Hubbs, 1936) was sister to Floridichthys Hubbs, 1926 , while Jordanella floridae Goode & Bean, 1879 was sister to Megupsilon Miller and Walters, 1972 – Cyprinodon Lacépède, 1803 ; and (iii) Lucania Girard, 1860 was sister to Wileyichthys Ghedotti & Davis, 2013 ( Fundulus lima Vaillant, 1894 – Fundulus parvipinnis Girard, 1856 ).

In our phylogeny, the families Cubanichthyidae, Cyprinodontidae , Fundulidae , Goodeidae , and Profundulidae were recovered as composing a monophyletic group, in agreement with recent studies ( Ghezelayagh et al. 2022, Piller et al. 2022). The branching pattern among families in this clade was in agreement with genomic results provided by Ghezelayagh et al. (2022) and Piller et al. (2022). However, in contrast to their and our findings, which place Cubanichthyidae sister to Goodeidae – Profundulidae, Piller et al. (2022) found that Cubanichthyidae was paraphyletic, with Cubanichthys cubensis (Eigenmann, 1903) sister to Cyprinodontidae , and with Cubanichthys pengelleyi (Fowler, 1930) sister to C. cubensis – Cyprinodontidae . Confidence intervals for divergence estimates of family originations in our tree (nodes 1–4; Table 2 View Table 2 ) overlapped broadly with confidence intervals for the same nodes in the study by Ghezelayagh et al. (2022). The greatest difference was for node 4 (separation of Goodeidae and Profundulidae ), wherein our confidence intervals overlapped with theirs, but their range also extended into more recent time, with their point estimate of ~22 Mya being substantially younger than ours of ~30.0 Mya.

Diversification through time

The lineage-through-time plots ( Fig. 4 View Figure 4 ) indicate that Cyprinodontidae is the only family that experienced a diversification-rate shift, with acceleration initiating ~10.9 Mya and continuing to the present (λ = 0.23 mean; range = 0.14– 0.38). The initial rate increase occurred when Megupsilon diverged from Cyprinodon . No shifts were detected in Fundulidae (λ =.12 mean; range =.07–.21), Goodeidae (λ = mean.13; range =.09–.29), or Profundulidae (λ = mean.12; range =.07–.24).

Reconstruction of ancestral areas

The best-fitting model for reconstruction of ancestral areas was the BAYAREALIKE+J (Supporting Information, Table S3 View Table 3 ), consistent with the coarse level of analysis, which recognized only the Nearctic and Neotropical realms. Most divergence events occurred within one realm or the other, as required by the BAYAREALIKE model (Garcia-R and Matzke 2021). Inclusion of the +J portion of the model implies that immigrations between realms occurred via long-distance dispersal ( Matzke 2014).

The ancestral distribution for this clade of North American killifishes is optimized as Nearctic–Neotropical, suggesting a widespread most recent common ancestor (MRCA) ( Fig. 5 View Figure 5 ). This clade first split into a northern fork with primarily Nearctic affinities ( Fundulidae – Cyprinodontidae ) and a southern fork with Nearctic–Neotropical affiliation (Cubanichthyidae, Profundulidae , and Goodeidae ). Appearance of fundulids in the Neotropics is relatively recent (Late Neogene–Quaternary), limited to the Fundulus grandis Baird and Girard, 1853 species group. Nearctic Cyprinodontidae made several Neotropical invasions ( Floridichthys , Yucatán Cyprinodon, Caribbean Cyprinodon , and Cyprinodon variegatus Lacépède, 1803 species group). The MRCAs of Cubanichthyidae and Profundulidae were Neotropical, and both families are restricted to that realm. Goodeidae is most likely to have had a Nearctic origin, with representatives of Girardinichthys , Allodontichthys Hubbs & Turner, 1939 , Xenotaenia Turner, 1946 , and Ilyodon reaching Neotropical drainages in the Late Neogene–Quaternary.

Reconstruction of ancestral habitats Ancestral-habitats reconstruction ( Fig. 2B View Figure 2 ) indicates that the MRCA of North American killifishes was coastal. Inland invasions within Fundulidae include Lucania interioris Hubbs & Miller, 1965 , the Fundulus sciadicus Cope, 1865 – Plancterus Garman, 1895 group, and later-branching lineages within subgenera Fundulus Lacépède, 1803 and Zygonectes Agassiz, 1854 . Fundulidae are the only family with species broadly distributed between coastal and upland habitats. In Cyprinodontidae , Floridichthys and Jordanella Goode & Bean, 1879 retained coastal affinities, while the maritime branch of Cyprinodon gave rise to upland and coastal lineages. Other branches within Cyprinodon diverged to become upland lineages. Cubanichthyidae remained coastal, whereas Profundulidae and Goodeidae are likely to have originated and diversified entirely in uplands. Overall, 58.6% of species had coastal distributions, 33.5% had upland distributions, and 7.9% had distributions spanning coastal and upland habitats ( Fig. 2B View Figure 2 ).

Biogeographical synthesis

Early branching of major lineages

Evidence suggests that the MRCA of North American killifishes arose in the Early Eocene Gulf of México ( Figs 3 View Figure 3 , 4 View Figure 4 ). Accordingly , the MRCA is likely to have had high salinity tolerance ( Ghedotti and Davis 2013). This timing corresponds precisely with a period of increased taxonomic and morphological diversification ( Ghezelayagh et al. 2022). The distribution of the MRCA potentially spanned the present-day Nearctic and Neotropical realms ( Fig. 5 View Figure 5 ). However , it was likely to be tropical because at this time; the tropics extended to 27°N, 3° north of the present-day boundary of the tropics ( Zhang et al. 2019).

The primary phylogenetic split in North American killifishes separated the northern sister families Cyprinodontidae – Fundulidae from a southern subclade of Cubanichthyidae– Goodeidae – Profundulidae 58.6–45.8 Mya ( Fig. 3 View Figure 3 , node 1; Table 2 View Table 2 ). The relative distributions of these families ( Fig. 1 View Figure 1 ), our ancestral-areas reconstruction ( Fig. 5 View Figure 5 ), and our ancestral-habitats reconstruction ( Fig. 2 View Figure 2 ) combine to suggest that separation occurred as a north–south division within the Gulf of México. The Tropic of Cancer was gradually retracting southwards through this period ( Zhang et al. 2019), which could imply that distinct subtropical ecosystems developed in the northern Gulf. We hypothesize that the northern MRCA of Fundulidae – Cyprinodontidae adapted to inhabit subtropical estuaries, including those of the Mississippi, Brazos, and Río Grande ( Fig. 6 View Figure 6 ). Concurrently, the southern MRCA of Cubanichthyidae– Goodeidae – Profundulidae potentially occupied a tropical estuary in the southwestern Gulf (Bejuco, Chicontepec, and Nautla) ( Fig. 6 View Figure 6 ). Geological evidence suggests the Gulf of México became isolated from the world ocean 55.8–55.0 Mya, accompanied by sea-level recession of 900– 1300 m ( Cossey et al. 2016, 2021). Because sea-level fall isolates estuaries ( Dolby et al. 2020), this extreme drawdown could have caused strong separation of northern and southern clades.

We hypothesize divergence of Cyprinodontidae from Fundulidae , 54.8–37.6 Mya ( Fig. 3 View Figure 3 , node 2; Table 2 View Table 2 ) was via east–west vicariance along the northern Gulf Coast. Drainage reconfiguration occurred in the northern Gulf of México 48.2 Mya (Early–Middle Eocene transition), when reduced sediment supply caused alluvial deltas to retract and separate ( Snedden and Galloway 2019). Shifting delta locations ( Snedden and Galloway 2019) might have initially dispersed and then subdivided killifish populations. To the northeast, the MRCA of Fundulidae was potentially isolated in the Middle Eocene Brazos River delta. From there, it is likely to have ranged east into the adjacent Mississippi Embayment, which flooded at this time ( Fig. 6 View Figure 6 ). The embayment refilled gradually through the Middle Eocene ( Snedden and Galloway 2019), suggesting that it contained extensive estuarine habitats that could have been the cradle for Fundulidae . To the southwest, the MRCA of Cyprinodontidae putatively originated in a separate delta region associated with the Río Grande and Río Bravo deltas ( Fig. 6 View Figure 6 ) that were comparatively sediment rich and wave swept ( Snedden and Galloway 2019). Such habitat is seemingly well suited for the ancestor of this group, which is hypothesized to have resembled Cyprinodon variegatus ( Echelle & Echelle, 2020) , a species well adapted for open coasts with fluctuating temperatures and salinities ( Simpson and Gunter 1956, Martin 1968, Nordlie 2006). This period also included ~ 40 m sea-level fall between 48.0 and 47.5 Mya ( Miller et al. 2020a, 2024), which is likely to have isolated each delta region ( Dolby et al. 2020). Subsequent sea-level recessions 46.9 and 45.6 Mya ( Miller et al. 2020a) might have amplified barriers to dispersal.

Divergence of Cubanichthyidae from Goodeidae-Profundulidae , 54.8–35.8 Mya ( Fig. 3 View Figure 3 , node 3; Table 2 View Table 2 ), could also be linked to sea-level fluctuations. We hypothesize that maximum sea levels associated with ice-free conditions in the Early Eocene, including a peak at 48.0 Mya ( Miller et al. 2024), allowed an ancestral taxon to range across the Yucatán platform, into the Caribbean Sea. If so, then subsequent sea-level falls in the Middle Eocene, along with failure to return to ice-free conditions until the end of the Eocene ( Miller et al. 2024), could have made the Yucatán platform a more formidable barrier, isolating the ancestor of Cubanichthyidae in the Caribbean ( Fig. 6 View Figure 6 ). However, as already noted, phylogenetic placement of Cubanichthyidae is inconsistent among recent studies (e.g. Ghezelayagh et al. 2022, Piller et al. 2022), indicating that this family requires additional study.

Divergence of Goodeidae from Profundulidae , 39.2–21.4 Mya ( Fig. 3 View Figure 3 , node 4), presumably reflects upland isolation of lineages in separate river drainages ( Table 2 View Table 2 ; Fig. 3 View Figure 3 ). We hypothesize that Goodeidae descend from a population that ranged inland into a Madrean River ( Fig. 6 View Figure 6 ), which joined the Bejuco delta system in the Early Eocene, after uplift of the Tamaulipas Arch diverted it southwards ( Snedden and Galloway 2019). As the Madrean River drainage coalesced among emerging tectonic basins and colliding terranes of southwestern North America ( Lawton et al. 2009), not necessarily incorporating any pre-existing rivers, this drainage might have been depauperate in freshwater fishes or lacked them entirely. If so, the Madrean River system might have provided open niches for coastal killifishes transitioning into freshwater habitats. In the Middle–Late Eocene, the outlet of the Madrean River shifted between the Tamaulipas Diversion and Río Bravo delta, but at the end of the Eocene (33.9 Mya) the Río Grande captured the inland drainage ( Galloway et al. 2011) ( Fig. 7 View Figure 7 ). A resulting influx of freshwater fishes might have constrained the distributions of goodeids to more marginal habitats isolated by distance upstream, barriers, or habitat conditions (i.e. salinity), presumably isolating upland populations from coastal relatives. For Profundulidae , the timing of divergence corresponds with uplift of the Chiapas Massif (40–25 Mya) ( Villagómez and Pindell 2020). Drainage from the Massif to the Bay of Campeche was localized at this time, not reaching the Gulf of México basin ( Beltrán-Triviño et al. 2021, Villagómez et al. 2022), isolating inland populations ( Fig. 7 View Figure 7 ). Given that cyprinodontids evidently inhabited the southwestern Gulf by ~31.8 Mya (see below; Table 2 View Table 2 ; Fig. 7 View Figure 7 ), we cannot rule out the possibility that a southward cyprinodontid invasion contributed to the disappearance of coastal Goodeidae – Profundulidae .