Conservation genetics of Cicindela deserticoloides, an endangered tiger beetle endemic to southeastern Spain. A.C. Diogo,1 A.P. Vogler,1,2* A. Gimenez,3 D.
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Conservation genetics of Cicindela deserticoloides, an endangered tiger beetle endemic to southeastern Spain A.C. Diogo,1 A.P. Vogler,1,2* A. Gimenez,3 D. Gallego3 and J. Galian4 1
The Natural History Museum, Department of Entomology, Cromwell Road, London, SW7 5BD, UK Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK 3 Department of Ecology and Hydrology, and 4 Department of Animal Biology, University of Murcia, 30017 Murcia, Spain 2
Received 12 August 1998; accepted 6 November 1998 Population surveys of the tiger beetle, Cicindela (Cephalota) deserticoloides, endemic to the few remaining salt steppes of southeastern Spain revealed only four extant colonies. DNA sequencing of some 1896 base pairs of mitochondrial DNA for one specimen each from three populations revealed only a single base pair change confined to a single of the three specimens, thus indicating an extremely low level of differentiation when compared to similar populations of Cicindela (s.l.) elsewhere. Divergence of C. deserticoloides from the closest relatives in the Iberian Peninsula was between 6.9 and 9.9%, attesting to the uniqueness of the species and its high conservation status. Habitat requirements appear to be phylogenetically conserved within Cephalota, but C. deserticoloides seems to be more narrowly confined to relatively drier conditions than its less endangered relatives. The geographic range of the relatives is wider and their local abundance higher, indicating that habitat specialization, low abundance and small geographic range in C. deserticoloides are correlated and in sum are responsible for its vulnerability to extinction. Keywords: mitochondrial DNA, insect conservation, Cicindelidae, Iberian biodiversity, rarity, salt flats
Introduction Many species of tiger beetles (genus Cicindela s.l.) are confined to saline soils where they inhabit isolated patches of habitat, frequently near temporal or permanent sources of open water. There is increasing pressure to utilize these areas for agriculture, leading to alteration of the water table and the chemical composition of the habitat. Because of the narrow habitat requirements of most tiger beetles (Pearson, 1988) these changes inevitably lead to the disappearance of specialized species. Several cases of local population extinctions have been documented and were shown to be directly correlated to anthropogenic habitat disturbance (Stamatov, 1972; Schultz, 1989; Knisley and Hill, 1992). In arid southeastern Spain several species in the genus Cicindela s.l. are confined to saline habitats in coastal and inland areas. One of these, C. (Cephalota) deserticoloides Codina (Codina, 1931), is a narrow endemic to the region around Murcia and Southern Alicante where it has been recorded only from very few locations in dry salt steppe habitat. Unfortunately, the unique salt steppe of southern Spain has been heavily disturbed, strongly
affecting C. deserticoloides. Localities for the species have a long history of being altered by drainage and desalination for transformation into agricultural land (Palao, 1909) and are now being used for industrial development or rubbish dumps, often accompanied by the construction of illegal buildings. The remaining populations are confined to isolated patches of suited habitat and have little or no contact, a situation that is aggravated by the increasing development and fragmentation of natural areas. These factors impact on C. deserticoloides throughout its entire geographic range and thus put the species in danger of extinction. For an effective conservation of C. deserticoloides, more information is needed to establish the current distribution and the level of threat. We also require improved taxonomic information on C. deserticoloides, in particular with respect to two issues. First, C. deserticoloides occurs in disjunct populations, which may be recognizably distinct genetically and therefore require separate management. Second, C. deserticoloides cooccurs with several morphologically similar species, whose level of divergence is unclear. We have used DNA sequences to address these issues, as a basis for
* To whom correspondence should be addressed at: Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK.
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imity of Arneva. Historically, the Guadalentin and Segura valley was a huge area of salt steppes that is fragmented nowadays for agricultural development. The Rambla de Ajauque constitutes a compartmentalized ensemble of small salt steppes connected by temporary streams in a very arid landscape. Habitats at all localities where C. deserticoloides was found can be described as the characteristic salt steppes of arid or semi-arid regions of the Iberian Peninsula (Bernaldez, 1992). Steppes are flat areas with saline soil which experience little or no flooding (i.e. they are dry during most of the year) but are affected by saline subterranean waters. The vegetation typically consists of a shrub layer (Sarcocornia fructicosa, Saueda vera and Halocnemun strobilaceum in La Alcanara) and a chamaephyte layer (Frankenia corymbosa and several Lymonium species) but vegetation cover is less than 50% of the surface area (Esteve et al., 1995). At present, available habitat is reduced to very few isolated patches in both main regions of the geographic range. At Arneva and San Isidro de Albatera there are only a few patches of several hundred square meters which are very fragmented and altered. The locality at La Alcanara is the best preserved although it is also fragmented within the agricultural landscape. Some parts of the salt steppes (about 7 km2) are subject to cycles of culture-abandonment-secondary succession (Caballero et al., 1994). The patches of Ajauque are threatened by the rise of the phreatic level due to the establishment of recent irrigation in the watershed of the local aquifer. This led to the replacement of salt steppes by halophyte communities dominated by Phragmites australis, a habitat not suitable for C. deserticoloides.
evaluating conservation priorities and for designating scientifically sound conservation strategies for this highly endangered species and its unique habitat.
Survey of existing populations C. deserticoloides has recently been collected in four localities, and records from four additional localities are found in the literature (Table 1). The four sites where beetles are currently known to occur have only been discovered in recent years after thorough surveys of potential habitats (Gallego et al., 1997) Gimenez, unpublished). Given the extent of these surveys it is unlikely that more sites sustaining substantial populations of the species will be found. It is also unlikely that the sites reported in the literature account for additional populations of C. deserticoloides. Two of the historical sites, Elche and Santa Pola, were surveyed by us many times, but beetles were never encountered. The record from Albatera, the type locality, could never be confirmed in our surveys; the record may actually pertain to our location at San Isidro de Albatera, some five kilometres south of Albatera. Finally, the reported occurrence at Totana (Sauleda, 1985) is about 1 km north of the current site at La Alcanara, but both places are part of the large salt steppe of the area TotanaAlhama and they may therefore not represent independent records. All current and historical sites of C. deserticoloides are confined to two (ecologically) distinct regions (Fig. 1). One is along a 100 km stretch in the Guadalentin and Segura valleys including the localities of La Alcanara in the west, Albatera and Arneva in the middle of the valley and Elche and Santa Pola in the east. The second area is the Rambla de Ajauque, a temporary stream tributary of the Segura river in the prox-
Table 1. Historical and present localities for C. deserticoloides. Asterisks indicate the extant localities. LA, SA, and RA are the population codes for the three specimens of our DNA analysis Localities
References
*La Alcanara (Alhama, Murcia) ‘LA’ *Arneva (Orihuela, Alicante) *San Isidro de Albatera (Alicante) ‘SA’ *Rambla de Ajauque (Fortuna, Murcia) ‘RA’ Albatera (Alicante) Elche (Alicante) Santa Ploa (Alicante) Totana (Murcia)
Gallego et al. (1997) Gimenez (unpublished results) Ortiz et al. (1987); present paper Gallego et al. (1997) Codina (1931); Ortiz et al. (1987) Vives and Vives (1978) Vives and Vives (1978); Sauleda (1985) Sauleda (1985)
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Figure 1. Map of southeastern Spain showing locality records for C. deserticoloides. Only the occurrences at La Alcanara, San Isidro de Albatera and Rambla de Ajauque could be established in this study. Note that the area around the location at Rambla de Ajauque, although geographically close to the locations in the Guadalentin River basin, differs ecologically.
DNA sequencing
the taxonomic status of C. deserticoloides. The species was grouped in the genus Cephalota (Rivalier, 1950) which consists of some 15 species with mostly circummediterranean and central Asian distribution. At least three of them also occur in southeastern Spain, including C. litorea, C. circumdata and C. hispanica. These are fairly similar morphologically and in part co-occur with C. deserticoloides. Arguments for the conservation of C. deserticoloides would be stronger if it is clear that this species is a phylogenetically distinct component of the Iberian fauna. DNA analysis on C. deserticoloides was hampered by the small number of available specimens and the poor quality of alcohol-preserved samples. Only one specimen each from the three known populations could be used for DNA extraction and sequencing.
The DNA analyses were aimed at two issues: first, the four current populations apparently lack contact and therefore may represent significantly differentiated populations, especially those in the more distant Totana–La Alcanara region. Allopatric isolates within the commonly recognized species of Cicindela have frequently been found to be differentiated morphologically (Willis, 1967; Boyd and Rust, 1982; Acorn, 1991) and genetically (Vogler and DeSalle, 1993; Vogler et al., 1993). Similar findings in C. deserticoloides would be informative with regard to pre-disturbance dispersal patterns, but also may require that different groups have to be recognized for conservation management. Second, it will be important to establish the validity of 119
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Table 2. Pairwise sequence divergence for isolates of C. deserticoloides and closest relatives. Absolute number of differences below the diagonal, differences as a percentage of the total sequence above the diagonal. Note that the sequences for C. hispanica and T. deserticoloides RA are incomplete
1 LOPHYRA flexuosa 2 TAENIDIA circumdata 3 TAENIDIA litorea 4 CEPHALOTA hispanica 5 TAENIDIA desert SA 6 TAENIDIA desert RA 7 TAENIDIA desert LA
1
2
3
4
5
6
7
– 188 209 106 198 134 196
0.09942 – 142 89 166 102 164
0.11224 0.07622 – 99 185 111 182
0.08453 0.07097 0.07926 – 103 103 101
0.10526 0.08820 0.09936 0.08233 – 0 1
0.09079 0.06906 0.07645 0.08214 0.00000 – 0
0.10476 0.08761 0.09817 0.08119 0.00053 0.00000 –
litorea. That topology, and in particular the relatively distant position of C. hispanica in the comparison of pairwise distances is also supported by the traditional taxonomy based on male genitalic characters (Rivalier, 1950) where the species was grouped in a different subgenus (Cephalota s.str.) to the others (Taenidia).
Also included were the three other Iberian species of Cephalota, and Lophyra flexuosa as outgroups. We sequenced three regions of the mtDNA covering genes for 16S rRNA and adjacent ND1, the cytochrome oxidase subunit 3, and cytochrome b, using protocols and primers as described previously (Vogler and Welsh, 1997), for a total of 1896 base pairs per individual. C. deserticoloides from Rambla de Ajauque and one of the relatives, C. hispanica, could not be amplified for the cytochrome b gene, leaving these sequences some 411 nucleotides shorter than the remainder of the taxa. Pairwise distances between these species are given in Table 2. We found no differences between the three specimens of C. deserticoloides, except for a single base pair polymorphism in the cytochrome b gene that was detected immediately adjacent to the region routinely used (Vogler and Welsh, 1997) for phylogenetic analysis in Cicindela, but well detectable on the chromatographs very near to the primer binding sites. Whereas this position clearly distinguished the specimen from La Alcanara and San Isidro, we could not assess the specimen at Rambla de Ajauque because difficulty in amplifying the cytochrome b gene in this specimen. All C. deserticoloides were highly distinct from the other species included in the analysis, with a minimum of 6.9% sequence divergence differentiating C. deserticoloides from C. circumdata (Table 2). In a parsimony analysis (not shown), the phylogenetic position of C. deserticoloides relationships could not be fully resolved; three shortest trees of 449 steps were consistent with a position of C. deserticoloides as the sister to the three other species of Cephalota. If, however, the DNA sequence for cytochrome b were removed from the analysis the most parsimonious tree (lengths 307 steps) supports a position for C. deserticoloides as sister to C.
Differentiation of C. deserticoloides populations No tests of gene frequencies that could provide estimates of genetic subdivision and dispersal between the remaining sites can be done based on our study. However, our data are sufficient to address the question about the number of independent conservation groups in C. deserticoloides, and the question of whether the isolated occurrence in the Rambla de Ajauque represents a differentiated entity. Among the large number of base pairs sequenced, only a single nucleotide change was detected, separating a La Alcanara specimen from the specimen at San Isidro. (No data for this base position is available on the specimen from Rambla de Ajauque, see above.) This level of differentiation between haplotypes is extremely low when compared to intra-specific diversity in endangered tiger beetles in North America, such as C. (Habroscelimorpha) dorsalis (Vogler and DeSalle, 1993) and C. (Ellipsoptera) puritana (Vogler et al., 1993). These studies however included samples from a much larger geographic range; locally, population variation was found to be very low in these studies as well, with one or a few very similar haplotypes dominating in a particular geographic area. Therefore, C. deserticoloides appears to fit a pattern observed elsewhere in Holarctic Cicindela (s.l) which lack significant local variation in a limited geographic area. For conservation management it is important to 120
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in the other species groups. It has been proposed that a species is more valuable to conserving biotic diversity the more divergent it is from its closest relatives. This reasoning led authors to set conservation priorities based on the total amount of diversity that is unique to a particular species (feature diversity of Faith, 1992). MtDNA with its generally clock-like rate of mutation provides a good approximation for the amount of diversity contained in a lineage and its unique features. Accepting the rationale of conservation priorities based on phylogenetic distances, data on branch length in C. deserticoloides indeed strengthen the case for protection of this species. This conclusion, however, has to be considered in the light of incomplete sampling of the genus Cephalota. As suggested by its name, C. deserticoloides is similar in morphological features to C. deserticola, a species with wide geographic distribution in central Asia. The species is rare in collections (Werner, 1992), and very little is known about its current distribution and abundance. Inclusion of this species in the DNA analysis may change the conclusions about the level of divergence from the closest relative of C. deserticoloides and the uniqueness of its features. However, in the absence of any reliable data on C. deserticola, and given the fact that the nearest locations for that species are in Armenia and northern Iran, it is probably justified to consider the status of C. deserticoloides irrespective of the situation in the related species. Therefore C. deserticoloides should be considered a unique element of the Iberian fauna deserving high priority for conservation.
recognize those groups of individuals or populations that constitute independent gene pools, because such genetically independent groups have a key role in the evolutionary process which conservation programmes attempt to conserve (Vogler and DeSalle, 1994; Cracraft, 1997). Principally a single nucleotide polymorphism, such as the one separating the specimens from La Alcanara and San Isidro, can be diagnostic (sensu Cracraft, 1983) distinguishing all individuals in one population from those in another and indicating complete separation of two such groups. For example, the finding of a single base consistently differentiating all individuals of an isolated population of C. dorsalis from other populations was sufficient to indicate high conservation status as a last remnant of a larger assemblage now largely extinct (Vogler, 1998). Because only a single specimen from each population of C. deserticoloides was included in our analysis we cannot judge whether or not any of the three surviving populations should constitute such a separate entity, and it would be necessary to include further specimens for this determination. However, it is reasonable to assume that these two distinguishable haplotypes are not confined geographically, and if they were, the separation is shallow and certainly does not indicate deep evolutionary partitions. For management purposes, it is also important to keep in mind that tiger beetle species are frequently persisting in a network of habitat patches subject to succession and (natural) disturbance from which populations disappear. Therefore a minimum number of populations is required for the long-term persistence of species; with only very few populations of C. deserticoloides left, and in the absence of positive evidence for genetic subdivision, all populations should therefore be considered a single entity for conservation purposes. The analysis of additional specimens is unlikely to add anything to this conclusion.
The causes of endangerment Why is C. deserticoloides more endangered than its close relatives? Habitat requirements in Cicindela are evolutionarily conservative, and in many cases small groups of closely related species occur in very similar habitat (Vogler and Goldstein, 1997). This is also true for the species of Cephalota which occur associated with saline conditions, near water bodies and small lagoons (Table 3). Related species should therefore be similarly endangered which does not seem to be the case, but remains to be investigated in more detail. However, C. deserticoloides appears to be more limited in its habitat requirements than its congeners. Animal and plant communities associated with salt steppes are organized according to the ecological gradients determined by the water availability and salinity of the soil. Tiger beetle communities are organized according to the same gradient (Ganeshaiah and Belavadi, 1986; Hidalgo et al.,
Sequence divergence in closely related species of Cephalota While the different isolates within C. deserticoloides are little divergent, they exhibit substantial DNA divergence to their closest relatives in the genus Cephalota. Uncorrected levels of sequence divergence were between 6.9% and 9.9% (Table 2). Compared to other species-level phylogenies for similar subgroups within the genus Cicindela (s.l.) from North America, divergence between species ranged between 0.7% and 8.9% in the C. maritima group and 0.8% and 9.5% in the genus Ellipsoptera. Thus, taxa within Cephalota are generally at the upper level of those found between species 121
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Table 3. Iberian species in the genus Cephalota, their taxonomic affinities and their distributional range. Data on distribution and habitat from Werner (1992) Species
Subgenus
Distribution
Range
Habitat
C. hispanica
Cephalota
southern Iberia
narrow
brackish water
C. litorea
Taenidia
circum-Mediterranean, Red Sea, Sudan, Somalia
widespread
halophilous, sea and lakes
C. circumdata
Taenidia
circum-Mediterranean
widespread
saline riparian
C. deserticoloides
Taenidia
southern Spain
narrow
dry saline areas
Implications for regional conservation management
1995). Although there are no detailed studies about habitat selection in C. deserticoloides its requirements seem to be very stringent. While co-occurring tiger beetles species such as C. litorea and Megacephala euphratica also select habitats where the soil is wet or even inundated for longer periods of time, the preferred habitat of C. deserticoloides is narrowly limited to the dry parts of the area. This fact could account for differences in the degree of endangerment between close relatives possibly because dryer locations are less common or may be more affected by current development. A second factor determining the degree of endangerment is the level of rarity, both with regard to local abundance and the extent of the total geographic range. Although we have not made an accurate estimation of population size, C. deserticoloides populations seem to be small compared to related species such as C. litorea which is more widely distributed and was consistently found in higher numbers. The spatial matrix in which populations exist depends on the availability of suitable saline flats in the area, which also strongly affect the overall abundance and metapopulation structure. Even before widespread development in the region, C. deserticoloides has been limited to a small set of locations, indicating either narrow habitat requirements not permitting the use of additional sites, or poor dispersal capabilities. Both would affect the overall abundance of this species and could result in short persistence times at a given location and low chances of recolonization. This could also affect the extent of the total distributional range. In contrast to C. litorea and C. circumdata with their wide distribution throughout the Mediterranean and Asia (Table 3), C. deserticoloides is a narrow endemic of southeastern Spain. It is possible that factors causing low local abundance are reflected also in the small global range of the species, as the tendency to extinction on a local level could translate into more limited global distributions.
Because of their narrow ecological requirements cicindelids are excellent taxa to monitor environmental changes and degradation affecting their habitats (Pearson and Cassola, 1992). The current decline of the cicindelid fauna in the salt flats of southeastern Spain certainly reflects vulnerability of other components of this unique ecosystem, including plants, birds and other insects. The presence of the endemic C. deserticoloides might indicate the existence of other endemic forms in this region, such as the tenebrionid Alphasida lorcana. C. deserticoloides is therefore a useful focal taxon for conservation, indicative of a relict assemblage from a formerly much wider distribution that was apparently only able to persist in this particular region. Protection of the remaining locations of C. deserticoloides is therefore of high priority and will also be useful in protecting a larger assemblage of genetically unique components. This has been recognized by the authorities, and the salt steppes of La Alcanara and Ajauque are designated protected areas by a Murcian regional law (4/1992; BORM). Unfortunately, their protection is not currently enforced.
Acknowledgements We thank J. Serrano, M.A. Esteve and J.L. Lencina for comments and locality information. Grant support by Portuguese JNICT (to ACD), British NERC (grant GR3/ 10632, to APV) and Spanish DGICYT (PB95–1005, to JG) is gratefully acknowledged.
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