Article Analyses of Genetic Diversity, Differentiation and Geographic Origin of Natural Provenances and Land Races of Casuarina equisetifolia Based on EST-SSR Markers 1, 2, 1, 1 1 1 Yong Zhang y, Pan Hu y, Chonglu Zhong *, Yongcheng Wei , Jingxiang Meng , Zhen Li , Khongsak Pinyopusarerk 3 and David Bush 3,* 1 Research Institute of Tropical Forestry, Chinese Academy of Forestry, Longdong, Guangzhou 510520, China; [email protected] (Y.Z.); [email protected] (Y.W.); [email protected] (J.M.); [email protected] (Z.L.) 2 Experimental Centre of Forestry in North China, Chinese Academy of Forestry, Beijing 102300, China; [email protected] 3 CSIRO Australian Tree Seed Centre, GPO Box 1600, Canberra, ACT 2601, Australia; [email protected] * Correspondence: [email protected] (C.Z.); [email protected] (D.B.) Co-first author. y Received: 13 February 2020; Accepted: 8 April 2020; Published: 10 April 2020 Abstract: Research Highlights: High variation of genetic diversity and differentiation among 27 seed sources within 14 natural provenances and 13 land race samples of Casuarina equisetifolia were found. High proportions of monoecious individuals may be present in some populations, as indicated by severe heterozytote deficiency and inbreeding found in many provenances and land races. The most probable origins of the land races were inferred according to the values of pairwise provenance differentiation and Nei’s genetic distances. Targeted introductions and testing of unrelated new accessions of C. equisetifolia from the Pacific and Philippines was proposed to identify Ralstonia-resistant genotypes. Background and Objectives: Casuarina equisetifolia was introduced to China a hundred years ago and has become a critically important tree species in coastal protection since the 1950s. Despite its importance, patterns of genetic variation, genetic relationships among natural provenances and probable origins of the land races remain unresolved. This has become a concern in China where Ralstonia solanacearum bacterial wilt has devastated plantations that are known to be from a narrow genetic base that urgently needs to be broadened. Materials and Methods: Fourteen natural provenances from Australia, Pacific islands and Southeast Asia, and 13 land race samples from parts of Asia and Africa outside the natural range were genotyped using 13 SSR (Simple Sequence Repeats) markers to characterize their allelic variation and genetic relationship. Results: Significant genetic diversity and differentiation among 27 seed sources within 14 provenances and 13 land race samples of C. equisetifolia was indicated. Significant heterozygote deficiency and inbreeding was indicated for a number of provenances, perhaps indicating a high proportion of monoecious parents in these populations. The most probable origins of the land races of the introduced countries were suggested according to the values of pairwise provenance differentiation (FST) and Nei’s genetic distances. Conclusions: We found significant genetic diversity and genetic differentiation among seed sources of C. equisetifolia. While individual land races do not appear to lack diversity, we were able to infer the origins of some, allowing targeted introductions of unrelated material to be made. In the case of the Chinese land race, targeting and testing new accessions from the Pacific and the Philippines may be a good strategy to identify Ralstonia-resistant genotypes. Forests 2020, 11, 432; doi:10.3390/f11040432 www.mdpi.com/journal/forests Forests 2020, 11, 432 2 of 17 Keywords: provenance; land race; allelic diversity; genetic relationship; microsatellite marker; Forestsgeographic 2020, 11, x origin; FOR PEERRalstonia REVIEW solanacearum 2 of 20 1. Introduction 1. Introduction Casuarina equisetifolia ssp. equisetifolia (hereafter referred to as C. equisetifolia) belongs to the CasuarinaceaeCasuarinafamily equisetifolia and has ssp. a wideequisetifolia natural (hereafter occurrence, referred with the to mostas C. southerly equisetifolia part) belongs of the range to the in Casuarinaceaenorthern Australia, family throughoutand has a wide southern natural Thailand, occurrence Malaysia,, with the Indonesia, most southerly the Philippines, part of the Melanesia range in northernand Polynesia Australia, (Figure throughout1)[ 1]. C. equisetifolia southern Thailand,is a nitrogen-fixing Malaysia, tree Indonesia, of considerable the Philippines, social, economic Melanesia and andenvironmental Polynesia ( importanceFigure 1) [1] in. C tropical. equisetifolia and subtropical is a nitrogen regions-fixing of tree the world.of considerable Over two social, million economic hectares andof Casuarina environmentalplantation, importance most of in which tropical are andC. equisetifolia subtropical, have regions been of plantedthe world for. woodOver two production, million hcoastalectares shelterbelts, of Casuarina vegetation plantation, rehabilitation most of which and forare ornamentalC. equisetifolia purposes, have been around planted the world for wood [2]. It production,is an important coastal plantation shelterbelts, species vegetation in India rehabilitation where it has undergoneand for ornamental genetic improvement,purposes around and the an worldimportant [2]. It and is an a significantimportant plantation tree in the Pacific,species wherein India planting where it is has thought undergone to have genetic commenced improvement, around and2700 an years important before and present a significant time [3]. tree in the Pacific, where planting is thought to have commenced around 2700 years before present time [3]. Figure 1. Natural distribution of Casuarina equisetifolia. The range of subsp. equisetifolia is contained Figurewithin 1. the Natural dotted linedistribution [1]. of Casuarina equisetifolia. The range of subsp. equisetifolia is contained within the dotted line [1]. In southern China, C. equisetifolia plays a critically important role in coastal protection. An estimated 300,000In southern hectaresof China,Casuarina C. equisetifoliaplantations, plays predominantly a criticallyC. important equisetifolia ,role have in been coastal established protection over. An the estimatedpast 70 years 300,000 along coastlineshectares of fiveCasuarina provinces plantations, (Hainan, Guangxi, predominantly Guangdong, C. equisetifolia Fujian and, Zhijiang)have been [4]. establishedThe plantations over immediately the past 70 years adjacent along to thecoastlines coast are of typically five provinces not harvested (Hainan, but Guangxi, left intact Guangdong, more-or-less Fujianpermanently and Zhijiang) for environmental [4]. The plantations amelioration immediately and protection adjacent functions. to the coast Inland are oftypically the coastal not harvested protection butzones, left which intact are more typically-or-les abouts permanently 200 m wide, for aenvironmental second belt, that amelioration is periodically and harvested, protection is establishedfunctions. Inlandin some of regions. the coastal The protection two contiguous zones, planting which are belts typically are typically about established 200 m wide, using a second the same belt, planting that is periodicallystock at any harvested, given location. is established in some regions. The two contiguous planting belts are typically establishedAlmost using 90% the of the same plantations planting stock established at any overgiven the location. past 30 years are clonal [5]. However, the geneticAlmost base 90% of these of the plantations plantations is veryestablished narrow, over with the only past 22 3 clones0 years identified are clonal from [5]. However, plantations the of geneticcoastal baseCasuarina of theseshelterbelts plantations of is Guangdong, very narrow Hainan, with only and 22 Fujian clones provinces, identified many from ofplantations which were of coastalthemselves Casuarina closely shelterbelts related [6]. of This Guangdong, is of particular Hainan concern and Fujian given provinces, that the shelterbelt many of plantingswhich were are themselvesrequired to closely remain related resilient [6] to. This both is biotic of particular and abiotic concern stressors. given In that recent the years,shelterbelt disease, plantings especially are requiredbacterial to wilt remain caused resilient by Ralstonia to both solanacearum biotic and [abiotic7], and stressors insect pests. In [recent8] have years, been disease a serious, especially problem. bacterial wilt caused by Ralstonia solanacearum [7], and insect pests [8] have been a serious problem. They have caused extensive mortality, resulting in declining productivity in those plantations that are harvested and reduced protection from wind and erosion in littoral shelterbelts. The effects of climate change, resulting in stronger winds, together with the need to plant on degraded coastal and inland sites with saline, alkaline, waterlogged and drought-susceptible soils also require a highly Forests 2020, 11, 432 3 of 17 They have caused extensive mortality, resulting in declining productivity in those plantations that are harvested and reduced protection from wind and erosion in littoral shelterbelts. The effects of climate change, resulting in stronger winds, together with the need to plant on degraded coastal and inland sites with saline, alkaline, waterlogged and drought-susceptible soils also require a highly resilient breed of tree with sufficient diversity to provide adaptation to these stressors. Clearly, the widely-used set of 22 clones is less than ideal in this regard. There is now