The phylogenetic and population analyses clearly show that on La Palma there is strong phylogeographic structuring for Palmorchestia populations and that subterranean populations have multiple independent origins from surface ancestors. Divergent mitochondrial lineages are restricted to precise cave systems or surface ravines and regions. Analysis for all but two populations sharing the same mtDNA haplotype supported monophyletic clades and a DNA sequence variation that denoted an absence of current gene flow between populations; the exceptions were the BG and CG north-east surface closely placed localities. AMOVA confirmed this and showed that when grouping Palmorchestia populations by habitat type, the genetic variation between groups proved very low. However, variation was maximized when considering localities (caves and surface ravines) as distinct populations, except for the north-east cave and surface sampling sites that contain a single population. This, added to the lack of reciprocal monophyly of P. epigaea and P. hypogaea mtDNA sequences, suggests that any division of the two species by habitat and ecology is unnatural.
Nuclear histone H3 gene and mitochondrial sequences showed the close relationship of Palmorchestia species with the Orchestia sp. of Gran Canaria. The two Palmorchestia species currently considered are clearly paraphyletic and genetically quite distant from the other Canary endemic landhoppers of the genus Orchestia. Within the Palmorchestia lineage three mitochondrial clades were obtained. The relationship of these mtDNA lineages with geography and geological history requires an understanding of the volcanic evolution of the island. The geological development of La Palma has been studied in detail . Two defined edifices can be distinguished ; the northern shield began to emerge over the sea at about 1.7–2.0 MYA and has undergone several volcanic cycles and terrain collapses that ended about 0.2 MYA. The southern ridge of the island is the product of much more recent and intense volcanic activity, beginning 0.12 MYA and lasting until the present. Thus, the southern half of La Palma, presumably including the shallow subsoil, is dominated by recent lava flows, some of them of in historic times. Only one of the three major mtDNA lineages within Palmorchestia is exclusive of hypogean populations, including a central and relatively more ancient lava tube (AR) and two southern younger caves (RA and MA). The other two lineages include surface and cave populations, one lineage from the north (AV, FR, GA) and the other from the north-east localities (CG, BG and BU) on the older northern shield, but surprisingly showing lower genetic divergences than the other lineages. The third mtDNA lineage includes south-west (PA), north-west (JA) and west (AG) populations. This shows that intraspecific genetic variation is clearly not directly linked to subterranean or surface habitats and that its geographic distribution cannot be explained only by the geological history of the island. In a phylogeographic study of the La Palma weevil Brachyderes rugatus, initial predictions based on the geological history of the island also proved to be too simple to explain the phylogeographic history of the species .
Divergence time estimations gave a faster molecular rate than other calculations made for stygobiont crustaceans using cytochrome oxidase subunit 1 (i.e. 1.25 × 10-8 substitutions per lineage per position per year ). Faster rates have nevertheless been suggested for subterranean Australian amphipods . However, mutation rates could be slower in subterranean organisms owing to longer generation times . Cave-dwelling organisms have lower metabolic, growth, fecundity and fertility rates and thus longer generation times than their surface relatives [31, 32]. Stygobiont amphipods and isopods need 8–10 years to reach sexual maturity, whereas their surface relatives only required 1–2 years [32, 33]. Accordingly, 8 years was assumed as the generation time for Australian stygobiont amphipods of the genus Pilbarus and Chydaekata . Similarly, in decapod crustaceans, generation times of 2 and 10 years for surface and subterranean closely related species, respectively, have been used , based on data published elsewhere [34, 35]. In addition, it has been debated whether there is a relationship between the rate of molecular evolution and sampling time [36–38]. However, several factors other than intrinsic problems with lineage rate variation and branch length estimation can produce overestimations of the molecular rate when dating nodes based on island ages . These include uncertainty of K-Ar dating caused by the burial of earlier lava flows resulting in underestimation of island age, population genetic variation within the ancestral island population and lineage extinction . We have obtained a reasonably good sampling of La Palma populations but there is a long branch in the phylogenetic tree joining the MRCA node of the sampled Palmorchestia populations and the sister species occurring in Gran Canaria, making it feasible that extinction could be a factor that has inflated our rate estimate. Nevertheless, mean time estimates for epi-hypogean transitions (or the contrary if we assume habitat reversion as a possibility) for two supported nodes in the Bayesian mitochondrial tree that relate surface and subterranean populations ranged from 0.36 to 0.94 MYA in the northern and western lineages, respectively. This suggests that independent episodes of colonization of the underground from surface pre-diverged lineages have probably occurred repeatedly in La Palma at different times during the Pleistocene and are probably related to documented drought episodes in the Canaries .
According to Stock's hypothesis , one can speculate on the role played by the relative drought of the island in the local extinction of surface Palmorchestia populations, except in permanently humid zones such as the scattered ravines radiating down the volcano (in particular, on the northern slopes exposed to north-east trade winds). In relation to this, two general competing hypotheses have been proposed to explain the transition from surface to subterranean life: the 'climatic relict hypothesis' (CRH)  and the 'adaptive shift hypothesis' (ASH) [39–41]. In the CRH, species pre-adapted to the cave environment (i.e. living in leaf litter or under stones) invade the hypogean habitat, with the epigean populations becoming extinct because of subsequent climatic change. In contrast, the ASH assumes active colonization of caves and parapatric speciation accompanied by adaptive differentiation and reduced gene flow between the epigean and hypogean populations. Support for both hypotheses has been obtained using mitochondrial phylogenies and molecular clock approaches (i.e. [9, 42]), although the lack of likely surface ancestral lineages and robust phylogenies makes it difficult to test them in particular cases . The pattern obtained in Palmorchestia suggests a recurrent entrance into the cave systems by already highly structured surface populations with subsequent independent adaptation to the hypogean environment and eventual interruption of gene flow between the two habitats. Local extinction of surface populations as the climate became drier  and multiple invasions of the underground make the pattern more consistent with the CRH than with a parapatric speciation via adaptive shift. The apparent contradiction between the age of the surface lava flows and some of the mtDNA lineages deduced by molecular clock approaches would support this. No surface populations have been found in the southern ridge of La Palma, but caves in this area are home to Palmorchestia ancestral haplotypes that could have derived from recent subterranean dispersion from north-central island populations. Indeed, some of the surface lava flows above these caves are recent but sampling shows ancient mtDNA haplotypes with estimated coalescent times of about 100,000 years, whereas another cave (Cueva de la Machacadora) harbours divergent lineages. If the lava tubes in these young island regions were coetaneous to surface lava flows, landhopper populations in these systems should be considered as recent newcomers and, at least in some cases, the product of independent colonizations. However, the sampling scheme has probably not covered all of the existing populations, including only a partial representation of the underground biodiversity, as access to this habitat is limited to the known entrances to the lava tubes accessible to humans (usually produced by roof collapse). In contrast, cavehoppers can easily disperse through cracks and crevices of the lava flows (the mesocaverns ), quickly colonizing the new cave systems underneath older terrains, either epigean or hypogean. This renders a scenario in which episodic colonizations involved dispersal from the surface to the underground, followed by recurrent range expansion and colonization through the subsoil, whereas on the surface isolation was caused by distance and fragmentation because of the patchy nature of the suitable habitat.
Eight independent networks were obtained using statistical parsimony (Figure 3). Five of these were site-specific and separated by many mutational steps, implying long periods of independent evolution. The north-east Palmorchestia mitochondrial lineage, which includes several surface populations and two lava tube populations, provides an opportunity to infer recent evolutionary events and relationships between lineages from the two habitats. This clade has an estimated age of 360,000 years using our relaxed molecular clock calibration (Figure 3 and Table 2), being the only clade that includes all mtDNA haplotypes from different populations into a single network under the limits of parsimony. Application of NCPA to this network showed an inference of contiguous range expansion from the northern surface (AV + FC) to north-east cave (BU) populations and previous gradual range expansion followed by fragmentation for the clade including populations BG + CG and the distant cave GA (Figure 4 and Table 4). Complex interactions either among populations within one habitat or among populations of the two habitats plus incomplete sampling and low sample size for some localities could partially explain why the significant associations found are few and dependent on the clade level (and thus the relative age of particular clades). In a similar study of the facultative cave-dwelling crayfish Cambarus tenebrosus, restricted gene flow and contiguous range expansion were the more frequent inferences, explaining the considerably and unusually large distribution of this species . This kind of pattern could occur frequently in populations of facultative subterranean organisms that have remained isolated for a sufficient time, but are lost or not evident in populations with long histories of isolation, as occurs in many of the Palmorchestia populations. Tests of association of haplotypes with habitat type or positioning in interior or terminal nodes in the network show that haplotypes are habitat-specific, but Palmorchestia has been present in both caves and surface habitats for a long evolutionary time. This can be deduced from the fact that ancient clades often occur in caves and in the network within the northern clade by the lack of statistical support for an association of surface haplotypes with interior nodes and of subterranean haplotypes with terminal nodes.
An added complication posed by cave fauna is the absence of morphological differentiation among divergent genetic lineages, resulting in the presence of cryptic species. This is in part caused by a high level of convergent evolution linked to the adaptation to darkness and has been shown recently for stygobiont amphipods [4, 7, 8]. In Palmorchestia, the independent evolution of populations for a considerable evolutionary time and fast molecular evolution are apparently uncoupled with morphological divergence. These results indicate that a re-evaluation of the taxonomic status of the current species of Palmorchestia is needed, with a revision of the presumed diagnostic morphological characters that differentiate P. epigaea and P. hypogaea and a search for characters that could identify individuals from different surface geographical regions and/or cave systems. If, as genetic data suggest, multiple entries to the subsoil have occurred in Palmorchestia, convergent morphological adaptation by eye degeneration and body depigmentation could have arisen independently by different mutations, as has been shown to occur in Astyanax fasciatus .