Conservation Genetics (2005) 6:213–226 Ó Springer 2005 DOI 10.1007/s10592-004-7830-x Increased inbreeding and inter-species gene flow in remnant populations of the rare Eucalyptus benthamii P.A. Butcher*, , A.K. Skinner & C.A. Gardiner CSIRO Forestry and Forest Products, GPO Box E4008, Kingston ACT 2602, Australia (*Corresponding author: Phone: +61 8 9480 3648; Fax: +61 8 9480 3641; E-mail: [email protected]; Current address: Kings Park and Botanic Garden, Fraser Avenue, West Perth WA 6005, Australia) Received 11 May 2004; accepted 1 August 2004 Key words: endangered species, Eucalyptus benthamii, genetic diversity, habitat fragmentation, hybridi- sation, mating system, microsatellites Abstract Eucalyptus benthamii Maiden & Cambage is a forest tree of interest for conservation and plantation forestry. It is vulnerable to extinction, occurring on the alluvial floodplains of the Nepean River and its tributaries, south-west of Sydney, Australia. These floodplains were largely cleared of native vegetation for agriculture by the mid-1800s. Flooding of the Cox Valley for Sydney’s water supply further decreased the species distribution. The species is now confined to one population of approximately 6500 trees in the Kedumba valley and three remnant populations on the Nepean River at Bents Basin (about 300 trees), Wallacia (nine trees) and Camden (about 30 trees). Genetic analysis of the four populations using microsatellite markers revealed significant divergence among all populations, despite the Bents Basin, Wallacia and Camden remnants being separated by distances of only a few kilometres. Trees in these populations have been estimated to range from 35 to 200 years old, suggesting genetic divergence among populations occurred prior to land clearing. To investigate the impact of fragmentation on the next generation, outcrossing rates were estimated from 41 families. While no direct relationship was found between population size and outcrossing rates, fragmentation and the isolation of trees appears to have resulted in higher levels of selfing and biparental inbreeding in seed collected from the Camden and Wallacia remnants. There was also evidence from seedling morphology that inter-species gene flow in- creased with fragmentation since 20% of the progeny from Camden and 30% of the progeny from Wallacia were hybrids. Seed viability and germination rates were significantly lower in the remnant populations, reducing their value as seed sources for regeneration and plantation forestry. To maintain the genetic integrity of the remnant populations, germplasm should be sourced from the local area. Outcrossed, non- hybrid seed could be produced by controlled pollination in ex-situ conservation stands or by using seedling morphology and microsatellites to screen seedlings from the remnant populations. Introduction population (Prober and Brown 1994). Long-term impacts of fragmentation can occur if increased Habitat fragmentation can have immediate and isolation alters patterns of gene flow, either by long-term effects on the abundance and diversity increasing gene flow within and among popula- of eucalypt ecosystems. The immediate effects re- tions (reviewed in Young et al. 1996), or reducing late to reductions in population size and declines gene flow in insect-pollinated species in genetic diversity, usually attributed to loss of (Cunningham 2000). Reduced gene flow may lead rare alleles as individuals are lost from the to increased inbreeding in eucalypts, most of which 214 have a mixed mating system. Adverse effects of inbreeding on seed production, viability, germi- nation and growth have been reported in several eucalypts (Hodgson 1976; Griffin and Cotterill 1988; Hardner and Potts 1995) and this may, in turn, threaten the long-term viability of small, fragmented populations. Camden white gum (Eucalyptus benthamii Maiden and Cambage) is a long-lived, tall forest tree with a restricted distribution that is considered vulnerable to extinction (NSW National Parks and Wildlife Service 2003). The species is confined to the alluvial banks of the Nepean River and its tributaries, south-west of Sydney, Australia. These fertile riverflats, favoured for agricultural devel- opment, were mostly cleared by the 1840s (Benson et al. 1996). The extent and size of populations of E. benthamii was further reduced following con- struction of Warragamba Dam for Sydney’s water supply and the associated flooding of the Cox Valley. Reductions in the species distribution, to- Figure 1. Geographical distribution of Eucalyptus benthamii. gether with interest in the species for plantation Filled circles show sampling sites; hatched areas indicate the forestry (Swain 2001) have focused attention on species distribution prior to land clearing (after Benson 1985). the need to conserve what remains of the species genetic resources (NSW National Parks and range of eucalypts, including E. deanei, E. crebra Wildlife Service 2003). F. Muell., E. punctata DC., E squamosa Dean & Eucalyptus benthamii is currently known from Maiden and E. acmenoides Schauer; exotic species two main sites; about 6550 individuals in the Ke- are uncommon. dumba Valley, Blue Mountains National Park and The major threat to E. benthamii is poor about 300 individuals at Bents Basin, part of regeneration due to competition with introduced which is protected in a State Recreation Area species and altered flooding and fire regimes. (NSW National Parks and Wildlife Service 2003). Additional threats include the impact of habitat In addition, approximately 30 scattered individu- fragmentation on genetic diversity; both direct ef- als occur along the Nepean River, near Camden fects due to the loss of alleles as the number of and nine trees downstream of Bents Basin near individuals in a population declines, and indirect Wallacia. The remnant populations near Camden effects due to reduced gene flow within and among and Wallacia consist of small groups of up to five populations leading to increased inbreeding. Most trees and isolated trees. Trees in the Camden eucalypts are insect-pollinated with a mixed mating population are dispersed along the banks of the system where inbreeding can lead to significant Nepean River over a distance of about 5 km, declines in growth and survival (see Butcher and adjacent to cleared agricultural land. The Bents Williams 2001 for review). Declines in diversity may Basin stand is a relatively dense, pure stand with also limit a species ability to respond to changing the understorey dominated by introduced weed selection pressures (Frankel et al. 1995), thereby species. It is separated from the remnant popula- limiting its long-term survival. Inter-species gene tions at Camden and Wallacia by about 5 km flow poses an additional threat to E. benthamii, (Figure 1). Many of the trees in the remnant particularly hybridisation with E. viminalis which populations are in poor health, and standing dead occurs naturally in the Camden region and readily trees are common. Other eucalypts occurring in hybridises with eucalypts that are in the same tax- the remnant populations include E. elata Dehnh., onomic section of the genus (Griffin et al. 1988). E. deanei Maiden and E. viminalis Labill. The In this study the level of genetic diversity in Kedumba population includes a more diverse Kedumba and the three remnant populations was 215 compared using microsatellite markers. Genetic numbers in the Camden and Wallacia remnants divergence among the populations was examined were limited by the number of trees with seed to determine whether the three remnants form crops. In Kedumba, sampled trees were separated distinct genetic entities. As the time since forest by at least 100 m to minimise sampling of related fragmentation is less than the lifespan of individuals. It was not possible to follow this E. benthamii trees, analysis of the adult trees is strategy in the remnant populations due to the likely to reveal more about patterns of diversity limited number of individuals with seed crops. before clearing. The impacts of fragmentation may Capsules from each tree were stored separately be more evident in subsequent generations if iso- in calico bags, dried at 35 °C for 2 days and seed lation of trees results in increased levels of extracted using a 600-lm sieve to separate seeds inbreeding with adverse effects on seed viability from chaff. The following measurements were and growth. Seed set and viability were therefore made on each capsule following methods described compared among populations. Outcrossing rates by Burrows (2000); weight of capsule contents were estimated from progeny arrays to provide an (seeds and chaff), number of seeds per capsule, indication of the possible consequences of frag- number of seeds per 10 g capsule contents and mentation on following generations. seed weight per capsule. To estimate seed viability, one to four replications of 50 seeds from each tree were sown into moist vermiculite in Petri dishes Methods and placed in growth cabinets at 25 °C for 2 weeks. The number of germinants per dish was Population sampling recorded after 2 weeks. Twelve seedlings from each of 41 trees were grown on and leaves sampled Mature leaves were collected from 16 trees in for the mating system analysis. Seedlings with leaf Camden, 17 trees in Bents Basin, nine trees at morphology typical of E. viminalis were recorded Wallacia and 32 trees in the Kedumba Valley as putative hybrids. Eucalyptus benthamii seedling population in December 2001. All trees in the leaves are ovate and pruinose (blue-green) and Wallacia remnant population were sampled. These differ from those of E. viminalis which are lance- were located in urban parkland between the river olate and green (Brooker and Kleinig 1999). and the township. The number of trees that could be sampled in the Camden population was limited by height of the crowns and concerns that sam- Microsatellite analysis pling using firearms or climbing would damage the trees which were located in an urban area. Twelve DNA was extracted from adult leaves using a trees in the Camden population were inaccessible. modified CTAB procedure, described by Glaubitz The location of populations and species distribu- et al.