minmize this pragraph:The data presented support a common role for climatic fluctuation, and the consequent fragmentation of forest habitat within South-East Asia (van der Kaars 1991; Kealhofer & Penny 1998; Penny 2001; Hope et al. 2004; White et al. 2004), in driving diversification and shaping the distribution of genetic diversity in nine species of Anopheles mosquito across the region. The best fitting demographic models for all six widespread species tested were those that included two allopatric refugia within the mountainous regions of northeast and northwest South-East Asia. With the exception of An. sawadwongporni, which may have increased in population size very recently, all species with sufficient sample sizes exhibited late Pleistocene (100–300 kya) demographic expansions, which are likely to have been triggered by the spread of forest habitat from refugia during warm, moist interglacial periods (Heaney 1991). Allopatric isolation within these refugia has presumably led to the simultaneous divergence of the eastern and western lineages of the CO2 gene observed in An. annularis, An. minimus, An. splendidus and An. philippinensis. This pattern of east–west divergence is also shared by An. baimaii and An. dirus, which have western and eastern distributions, respectively, although divergence in this case has resulted in speciation (Morgan et al. 2010). In contrast, no deep divergences were observed in An. aconitus, An. sawadwongporni or An. maculatus. Despite such different outcomes in patterns of genetic structure, the demographic modelling process was nonetheless able to detect a common influence of environmental history on the distribution of genetic diversity across all species.

Differences between species in the patterns of genetic diversity, which are present despite a common influence of Pleistocene environmental change, can be explained by a combination of stochastic effects and differences in species biology. The SPLATCHE modelling approach can provide some insight into the latter. The fit of real to simulated data sets was influenced by levels of pre-expansion migration between refugial regions, representing levels of gene flow as populations expanded from allopatric refugia during the interglacials. Whereas An. minimus and An. philippinensis best fitted a model of low pre-expansion migration, all remaining species best fitted a model with higher levels of gene flow, potentially representing differences in dispersal capacity.

The model fitting was also influenced by the breadth of altitudinal range of high carrying capacity and low friction during glacial periods, corresponding to a species’ tolerance to glacial habitats. Anopheles minimus and An. baimaii best fit models that have a low altitudinal range, consistent with our understanding that these species are highly forest dependent (Reid 1968; Darsie & Pradhan 1990; Rattanarithikul et al. 2006). Anopheles aconitus and An. sawadwongporni are the only species that best fit evolutionary scenarios with a wider altitudinal range, indicating greater flexibility to glacial habitats. Anopheles aconitus is more of an ecological generalist, utilizing exposed larval habitats such as rice paddies and ditches (Overgaard et al. 2003). During glacial periods, An. aconitus may have been able to survive in a relatively wide range of habitats, providing they retained sufficient moisture to provide appropriate larval habitat. A similar argument may apply to An. sawadongporni as its larval habitats, shaded streams (Rattanarithikul et al. 2006), could also potentially occur across a wider altitudinal range than forest habitats. However, it would then be surprising that An. maculatus, which is apparently ecologically similar and closely related to An. sawadongporni (Rattanarithikul et al. 2006; Morgan et al. 2009), best fitted models with narrow altitudinal ranges of suitable glacial habitat. This apparent difference is also striking given that over the same area of mainland South-East Asia (i.e. excluding eastern China, Taiwan and Sri Lanka), the gene trees of both species indicate recent range expansions. This suggests that the differences in the altitudinal ranges of suitable glacial habitat for these two species may be due to limitations in the SPLATCHE modelling approach, for example, in the limited number of scenarios modelled and/or the greater geographical sampling coverage for An. maculatus. If An. aconitus, An. sawadongporni and perhaps also An. maculatus are more plastic in their habitat requirements, this may underlie their lack of formation of deeply divergent lineages, presumably owing to lower levels of isolation during glacial periods compared to the other more forest-associated species in this study.

The spatially explicit modelling approach provided strong evidence for the persistence of mosquito populations in two allopatric northern refugia, in the east and the west of South-East Asia, during the Pleistocene glacial periods. The approach had insufficient power, however, to distinguish between the potential eastern regions of northern Vietnam and Yunnan, and between the potential western regions of northern Myanmar and northeastern India. Northern Vietnam, northeastern India and northern Myanmar were all identified as putative forest refugial regions, based on a phylogenetic study of the Neocellia Series of Anopheles mosquitoes (Morgan et al. 2009). Northern Vietnam has also been proposed as a refugial region for the following forest-associated taxa: Anopheles baimaii (Morgan et al. 2010), the bird species Sasia ochracea (Fuchs et al. 2008) and Colobinae monkeys (Brandon-Jones 1996). The substantial population structure within Anopheles jeyporiensis, which is limited to the north of South-East Asia, further supports the persistence of suitable forest habitat in the north throughout the Pleistocene. Increased, high-density sampling throughout the mountainous northern regions may enable the locations of Pleistocene forest refugia to be more precisely pinpointed, the relative importance of those refugia defined and any additional complexity, such as microrefugia (Rull 2010), identified.

Because of the long-term persistence of populations within glacial refugia, such regions are expected to accumulate greater diversity than recently colonized, less stable regions (Hewitt 1999, 2001). For example, within continental Europe, the long-term survival of many species in southern refugia has led to greater genetic diversity in the south relative to the more recently colonized north (Hewitt 2001, 2004). In the forest-dependent South-East Asian taxa studied here, a similar pattern is evident, but genetic diversity increases with increasing latitude. North–south clines of decreasing diversity along similar regions of the Thai–Myanmar border have also been noted at the intraspecific level in the black fly, Simulium tani (Pramual et al. 2005) and at the species level in sphingid moths (Beck et al. 2007). This commonality of latitudinal diversity gradient together with the demographic modelling supports a scenario of long-term survival in mountainous northern refugia followed by more recent colonization of the south for a range of taxa. All of the putative refugial areas, northeast India, northern Myanmar, Yunnan and northern Vietnam, are not only rich in genetic diversity as observed here but are also noted regions of high species biodiversity and endemicity (Pramual et al. 2005; Sterling & Hurley 2005; Ying-shan et al. 2007). The results of this study support the importance of montane regions as sources and sinks of tropical biodiversity, as has previously been documented in Borneo, Malaya and Sumatra (in Crematogaster ants; Quek et al. 2007), in Africa (Fjeldså & Lovett 1997; Roy 1997; Bowie et al. 2006) and in South America (Fjeldså & Rahbek 2006).

The Thai–Myanmar border is a suture zone where the deeply divergent eastern and western lineages of An. minimus and An. annularis overlap, and also a potential hybrid zone where the sister taxa An. dirus and An. baimaii meet (Morgan et al. 2010). Increased sampling within the region is necessary to determine whether An. splendidus and An. philippinensis also have suture zones along the Thai–Myanmar border. Simultaneous divergence of eastern and western lineages within An. annularis, An. minimus, An. philippinensis and An. splendidus is strongly supported by hABC analysis and estimated to have occurred within the mid-Pleistocene, ∼900 or ∼1300 kya, depending on the divergence rate used. The ∼900 kya estimate is likely to be more accurate because this used the divergence rate of 2.3% pmy (Brower 1994), which was estimated from groups taxonomically similar to Anopheles (e.g. several Diptera) and the same gene, CO2. Conversely, the 1.5% pmy divergence rate estimated by Quek et al. (2004) used a very broad range of arthropods (including crabs) and a different gene, CO1. Allopatric divergence in the speciation of An. dirus and An. baimaii also dates to a similar time period, ∼1 Ma using the faster Brower (1994) rate of divergence (Morgan et al. 2010). The simultaneous and apparently coincident formation of suture/hybrid zones across several species suggests that this divergence was indeed driven by a common influence of environmental change. The estimated date (using either molecular clock) is too recent to have been triggered by tectonic activity or orogony, which dates back to 5 Ma or earlier in this mainland region (Hall 2002). A more likely explanation for the initial driving of divergence is the change in Pleistocene glacial cycles approximately 1 Ma, such that the cycles occurred more rapidly and with increased severity (Imbrie et al. 1992). The increased environmental pressures resulting from this change may have intensified the fluctuations in forest cover, triggering diversification within forest-associated taxa.

The positioning of the suture zone may result from the southwards projection of the northern mountainous region into the narrow mountainous formation along the Thai–Myanmar border (see Fig. 1). At the transition into the post-glacial period, the regions close to the mountains are likely to have been reforested prior to the more distant and initially more arid areas of central Myanmar and Thailand. Consequently, the mountainous projection along the Thai–Myanmar border could facilitate early southwards dispersal from eastern and western refugia along its eastern and western flanks to form the suture zone.

If the common patterns in the distribution of genetic diversity detected in this study are also found in other forest-associated taxa, the refugial regions of the mountainous north of South-East Asia and the suture zone on the Thai–Myanmar border would have particularly high conservation value. These regions not only currently possess high and unique genetic diversity but would also be important for the generation and maintenance of biodiversity in the long term. Thus, northern Vietnam, Yunnan, northern Myanmar and northeastern India, as well as the border region of Thailand and Myanmar, are highlighted as important regions for further study. In this respect, Anopheles mosquitoes will be useful in more fine-scale studies of these regions to characterize in greater detail the effects of Pleistocene environmental change. Above all, it is imperative that comparable studies of other forest-dependent taxa, including vertebrates, are conducted to determine the taxonomic generality of the findings of this study and ensure that this understanding can be taken into account in planning conservation policy.