Sympatric lineages in the Mantidactylus ambreensis complex of Malagasy frogs originated allopatrically rather than by in-situ speciation.

• Two deep genetic lineages detected in the Malagasy frog, Mantidactylus ambreensis. • Co-occurrence without admixture confirms separate species status of these lineages. • The lineages are segregated by elevation. • Phylogeography suggests this co-occurrence originated by secondary contact. • No evidence for sympatric or parapatric speciation. Madagascar's biota is characterized by a high degree of microendemism at different taxonomic levels, but how colonization and in-situ speciation contribute to the assembly of local species communities has rarely been studied on this island. Here we analyze the phylogenetic relationships of riparian frogs of the Mantidactylus ambreensis species complex, which is distributed in the north of Madagascar and was originally described from Montagne d'Ambre, an isolated mountain of volcanic origin, currently protected within Montagne d'Ambre National Park (MANP). Data from mitochondrial DNA, and phylogenomic data from FrogCap, a sequence capture method, independently confirm that this species complex is monophyletic within the subgenus Ochthomantis , and identify two main clades within it. These two clades are separated by 5.6–6.8% pairwise distance in the mitochondrial 16S rRNA gene and co-occur in MANP, with one distributed at high elevations (940–1375 m a.s.l.) and the other at lower elevations (535–1010 m a.s.l.), but show almost no haplotype sharing in the nuclear RAG1 gene. This occurrence in syntopy without admixture confirms them as independent evolutionary lineages that merit recognition as separate species, and we here refer to them as high-elevation (HE) and low-elevation (LE) lineage; they will warrant taxonomic assessment to confidently assign the name ambreensis to one or the other. Populations of the M. ambreensis complex from elsewhere in northern Madagascar all belong to the LE lineage, although they do occur over a larger elevational range than in Montagne d'Ambre (285–1040 m a.s.l.). Within LE there are several phylogroups (LE1–LE4) of moderately deep divergence (1.5–2.8% in 16S), but phylogroup LE4 that occurs in MANP has a deeply nested phylogenetic position, as recovered separately by mitochondrial and sequence capture datasets. This suggests that HE and LE did not diverge by a local fission of lower and upper populations, but instead arose through a more complex biogeographic scenario. The branching pattern of phylogroups LE1–LE4 shows a clear south-to-north phylogeographic pattern. We derive from these results a testable hypothesis of vicariant speciation that restricted the HE lineage to MANP and the LE candidate species to a climatic refugium further south, with subsequent northwards range expansion and secondary colonization of MANP by LE. These results provide an example for complex assembly of local microendemic amphibian faunas on Madagascar. [ABSTRACT FROM AUTHOR]