Conservation of threatened plants of Crete: seed ecology, operation and management of a gene bank

The purpose of this study is to investigate the biology of seeds and the ex situ conservation of threatened plants of Crete. Specifically, the thesis aims at an ecophysiological approach of seed dormancy and germination and at addressing and understanding the problems arising from the operation of a Seed Bank of native plant species. The plants under consideration are 66 taxa of the Cretan flora. These plants have been selected since they are listed as threatened in the first publication of “The Red Data Book of Rare and Endangered Plants of Greece” (Phitos et al. 1995). Many of these are also protected by international conventions and laws. Moreover, the 66 taxa studied represent seven different life-forms, high phylogenetic diversity (51 genera and 28 families) and numerous habitats of Crete. Fifty of the taxa studied are endemic to Crete and eight are endemic to Greece, while the remaining eight have a wider geographical distribution in the S.E. Mediterranean region. The seed collections were made during the period 2000-2009. From the 66 target taxa, 62 have been located and seeds were collected from 61. A total of 135 collections have been made and the problems and difficulties encountered in their implementation are described and practical solutions are proposed. The collections of seeds from certain taxa were not sufficient to fully investigate the issues raised in this study due to unknown or improper collection periods, biological peculiarities of certain species, the inaccessibility of populations, disturbed habitats and small population size. Small collections have been renewed by cultivating plants in the Botanical Garden of MAICh and, recently, in the Alpine Garden of the Forest Directorate of Chania. In order to avoid hybridization among individuals of different populations, the cultivated individuals of each species represent only the population from which the original collection was made. The maturation and dispersal periods of seeds were identified for each studied taxon. The dispersal units in the cases where they are different from fruits or seeds, the mechanisms of dispersal (Astragalus idaeus, Carlina diae, Limonium creticum,) as well as the phenomenon of bradychory (Campanula laciniata and Silene holzmannii) are described. Potential problems of sexual propagation are discussed, the percentage of 'empty' seeds in collections has been determined and the masting behaviour of Zelkova abelicea is confirmed. The description of the external morphology of fruits and seeds contributes significantly to the identification of the species at the time of collection. The fruits and 418 seeds of all the collected taxa are described and the descriptions are supplemented with photographs and biometric data. In this study, fruits of 17 taxa and seeds of 14 taxa are described for the first time and additional information is given on the description of fruits of 2 taxa. Seed anatomy and embryo morphology are associated with certain classes of dormancy, and thus with the germination of seeds. The description of the embryos of mature seeds has been mainly based on the description of embryos as determined by the keys of Martin (1946) recently revised by Baskin C.C. & Baskin J.M. (2007). The embryos of 61 taxa are described and, overall, they do not differ from the typical embryos of each family as described in the literature. Only a few specific peculiarities were observed, such as the pseudomonocotyly trait in the endemic plant of Crete Horstrissea dolinicola of the monotypic genus Horstrissea. The state of seed dormancy upon harvest, i.e. the primary dormancy of seeds, was determined in 57 taxa according to the keys proposed by Baskin J.M. & Baskin C.C. (2004). The criteria for determining primary dormancy are discussed based on the problems encountered in their application and the following ammendments are proposed: 1) The period allowed for germination of non-dormant species should not be limited to 30 days (as in the case of Zelkova abelicea). 2) A redefinition of the method that correlates morphological dormancy with a small embryo within abundant endosperm is proposed (as in the case of Phoenix theophrasti). Approximately 50% of the seed collections are characterized as non dormant, while all major classes of primary dormancy (physiological, physical, morphological and morphophysiological) - except for combinational (physiological and physical) - are determined in the remaining 50% of the collections. The most prevalent class of dormancy identified is physiological dormancy (21% of the taxa studied). In addition, conditional dormancy (non-deep physiological dormancy) of the seeds was difficult to determine with certainty in 16% of the collections but, taking into account other data, these species are characterized as either exhibiting physiological dormancy (7%) or non dormant (9%). The release of primary physiological dormancy, especially in low-altitude species (0-1000 m), occurs during dry storage in laboratory conditions or in the dry room of the Seed Bank (a process known as after-ripening). Initial experiments have shown that cold stratification contributes to the release of physiological dormancy in dormant seeds of species from medium (1000-1800 m) and high altitude (>1800 m). The induction of secondary dormancy by unfavourable conditions of temperature and light (white light or darkness, depending on the taxon) was investigated in 23 taxa and imposition of secondary dormancy was observed in 6 of them. It was found that in 419 the annual taxon Silene integripetala subsp. greuteri, secondary dormancy is imposed by either high temperatures (thermodormancy) or continuous darkness (skotodormancy). In this plant, both primary and secondary dormancy are released by storing the seeds in the dry room. On the basis of these data, a pattern of the potential annual changes in the seed dormancy state (the so called „dormancy cycle‟) of S. integripetala subsp. greuteri in nature is put forward. Approximately 20% of the taxa exhibit morphological, morphophysiological and a special type of morphological dormancy. The morphological and morphophysiological types of dormancy are observed in taxa of the families Apiaceae and Ranunculaceae, which are characterized by linear - underdeveloped and rudimentary embryos, respectively. The special type of morphophysiological dormancy is observed in the species Cistanche phelypaea (Orobanchaceae) and Cephalanthera cucullata (Orchidaceae), which are characterized by undifferentiated embryos. The seeds of the pseudomonocotylous species Horstrissea dolinicola are characterized by double morphological dormancy. According to all the experimental evidence, the establishment of seedlings of this species in the wild is completed the second spring after dispersal of the seeds (seed dispersal takes place in autumn). Physical dormancy is shown to characterize 4% of the taxa (the species Astragalus idaeus and Convolvulus argyrothamnos). The seeds of these taxa are characterized by primary dormancy, imposed by the hard, water impermeable seed coat (physical dormancy). The weakening of the seed covering layers results in the removal of the physical dormancy of the seeds. The effect of constant temperatures (5)10-20(25, 30) °C on seed germination was studied in 43 taxa. The taxa are grouped according to the range of optimal germination temperatures: (5)10-15 °C (low temperature taxa), (5)10-20(25, 30) °C (broad-range temperature taxa), 15 °C (intermediate temperature taxa) and 15-20(25, 30) °C (high temperature taxa). In a large proportion of the taxa studied (about 40%), germination is favoured at low temperatures (typical Mediterranean taxa) and an almost equal percentage of taxa germinate at a wider temperature range (broad-range temperature taxa). Only 14% of the taxa „prefer‟ high temperatures. The main conclusions from the study of the role of temperature on germination are: 1) taxa that germinate at high temperatures either grow at high altitudes (alpine taxa) or belong to genera with a wider geographical distribution in the tropics, 2) high altitude taxa are either characterized as „broad temperature taxa‟ or germinate only at high temperatures, that is they do not germinate at low temperatures, and 3) two species (Androcymbium rechingeri and Alyssum sphacioticum) which are characterized by conditional dormancy germinate at a very narrow range of intermediate temperatures (around 15 °C). 420 The effect of white light on germination was studied at all temperatures and compared to the germination of the seeds in continuous darkness at the same temperatures. The duration of the experimental photoperiod per 24 hours was 12 h in each chamber. White light seems to inhibit the germination of a significant proportion of the taxa (46%) while it promotes germination in 35% of the taxa and does not affect germination in only 19% of them. The light used for the experimental study of germination in the growth chamber differs from sunlight both qualitatively and quantitatively and therefore the response of germination to light is indicative. The experimental evidence concerning the germination response to light has led to the following conclusions: 1) Datisca cannabina and Campanula hierapetrae, the two species showing an absolute light requirement for germination, are characterized by very small seeds. Promotion of germination by light occurs in other taxa with small seeds as well, such as all taxa studied within the genus Campanula, Hypericum jovis and others. Absolute requirement for light was also found in the alpine species Nepeta sphaciotica and the submerged aquatic plant Callitriche pulchra, 2) the inhibitory effect of white light on germination was observed (among others) in certain maritime taxa (e.g. Silene ammophila subsp. carpathae, S. succulenta subsp. succulenta) but also in two geophytes (Arum purpureospathum, Bellevalia brevipedicellata), and 3) photosensitivity in some cases depends on the germination temperature, that is the inhibitory or promotive effects of light are not exhibited in the entire temperature range that a taxon can germinate. According to the results of the study of dormancy and the effects of environmental factors on germination, the optimum conditions for germination were determined for 46 taxa. Additionally, a preliminary outline of germination behaviour has been obtained for another 15 taxa. With a few exceptions, seed germination of the threatened taxa of Crete has been investigated for the first time in the present thesis. The seed collections are grouped according to the season of collection. The seasons of collection are designated as follows: spring (April-June), summer (July- September), autumn (October-December). These seasons are not identical with the conventional seasons, but they represent: a) the wet-dry transition period, b) the period of intense drought, and c) the period of rainfall initiation, respectively, of the typical Mediterranean climate of Crete. Seed dormancy as well as the effects of temperature and light on germination are observed to be associated with time of collection. Dormancy is shown to be related with time of collection as follows: 1) the majority of taxa that grow at low altitude and mature their seeds in spring are characterized by primary physiological dormancy - about 50% are characterized by physiological dormancy while an additional 20% are probably characterized by physiological dormancy as well, 2) is not exhibited by low altitude taxa that mature 421 their seeds in summer or autumn; however, physiological dormancy is detected in high altitude taxa that mature their seeds in summer and in middle altitude taxa that mature their seeds in autumn, 3) the morphological and morphophysiological classes are the main classes of dormancy in the autumn collections, and 4) in low-altitude taxa, a seasonal increase of non-dormant (and possibly non-dormant) seeds is observed from in spring (26%) to summer (73%) and in autumn (86%). The temperature range of germination and the time of collection are observed to correlate as follows: 1) the majority of broad-range temperature taxa ripen their seeds in summer and 2) most taxa that 'prefer' low temperatures for germination ripen their seeds in spring and autumn, 64% and 60% respectively. The effect of light on germination and the season of collection are correlated as follows: 1) the inhibitory effect of light on germination is high in taxa that mature their seeds in spring and summer (71% and 47% respectively) and is significantly reduced in autumn (10%), 2) there is a gradual increase in the promotion of germination by light from 21% in spring collections to 31% in summer and 60% in autumn collections; the increase is mainly observed in taxa in which the promoting action of light depends on temperature (i.e. it is not displayed throughout the germination temperature range), and 3) the 'indifferent' response of germination to white light increases from spring to autumn collections (from 7% to 30%). The investigation of ecophysiology of dormancy and germination focused on identifying potential adaptive strategies shown by the threatened plants of Crete to ensure their survival; the main conclusions are summarized below: 1) Primary dormancy and the induction of secondary dormancy, photosensitivity, the temperature range of germination, bradychory, seed polymorphism (Tragopogon lassithicus) and myxospermy (Artemisia herba-alba) are observed and discussed in relation to habitat, life-form, seed size etc. Most findings confirm similar adaptations (usually on the generic level) from the existing relevant literature and seem to be related to adaptation strategies developed by typical Mediterranean plants; these strategies fine tune seed germination timing at favourable conditions for seedling emergence and offspring survival. 2) The detection of primary physiological dormancy in the laboratory and its release (by storage of seeds in the dry room – after-ripening) is believed to offer a new perspective to the study of the ecological role of physiological dormancy in Mediterranean plants, especially those grown in seasonally dry environment. It appears that the primary physiological dormancy prevails in taxa with seeds dispersed in spring while the dormancy is relieved in summer as the result of high temperatures and drought. Thus, non-deep physiological dormancy 'compels' seeds to germinate in autumn (rainy period) while at the same time „prevents‟ germination in springtime (pre422 drought), although the average climatic conditions (light, temperature, soil moisture) are similar in both seasons. 3) Certain characteristics of dormancy and germination of several taxa are considered „phylogenetically residual‟, not directly related to their adaptive strategies. For example: a) the „preference‟ for high temperatures of germination in the species Periploca angustifolia and Phoenix theophrasti (the genera of which show a wider geographical distribution in subtropical and tropical regions) and b) the morphological dormancy of seeds of Bupleurum gaudianum (Apiaceae). The storage behaviour of seeds has been investigated in 36 taxa according to the protocol proposed by Hong & Ellis (1996) while the storage behaviour for a number of additional taxa was indirectly determined by the type of fruit (Hong et al. 1998). All taxa studied are characterized by orthodox seed storage behaviour (tolerance of seed dehydration) and therefore they can be stored for long periods in a seed bank. Within the operation framework of the Seed Bank of MAICh, various factors were identified that affect seed germination behaviour of a population: 1) the time of collection, as different germination behaviour was observed among different collection dates, 2) the renewal of collections, since seeds of some taxa grown in the Botanical Gardens are less dormant than seeds from natural population (in cultivated plants of Asperula crassula and Crepis sibthorpiana a widening of the germination temperature range and a reduction of photosensitivity were observed), 3) storage of seeds in the dry room and in the laboratory, which significantly affected the germination behaviour of many taxa (in almost all taxa studied the germination rate was increased or not affected by storage, while only for the species Phoenix theophrasti a significant reduction of germination rate was observed), and 4) in most cases, the storage of seeds in the freezer (- 20 °C) of the Seed Bank for several months did not affect final germination percentages and germination rates of the seeds (only for the species Nepeta sphaciotica a decrease of germination rate was observed while for the species Phoenix theophrasti the germination rate increased after storage in the Seed Bank). This thesis contributes to: 1) the investigation of germination of Mediterranean plants and the possible adaptive strategies followed that „ensure their survival‟, 2) the optimization of the operation of Gene Banks of native species by providing empirical knowledge for the various, different procedures of a Seed Bank (collection, cleaning, drying, storage, renewal of collections) with genetic material of great diversity and of many different habitats and 3) the proposal of management and conservation measures for the protection of natural populations of the threatened plants of Crete

ISSR genotyping of Phoenix theophrasti natural populations in Turkey and Crete (Greece) and P. Dactylifera

Intraspecific and interspecific diversity between Phoenix theophrasti individuals (92 from Turkey and 70 from Crete, Greece) and P. dactylifera specimens (28 from Turkey) were investigated by inter-simple sequence repeat (ISSR) analysis. A total of 45 polymorphic fragments, 360–3454 bps long, were produced. Intraspecific diversity for P. dactylifera was 26.63% and similarities ranged between 0.5 and 1. In the constructed dendrogram, P. dactylifera specimens clustered together in the first main group, outside branches consisting of P. theophrasti samples that generated the second main group. The intraspecific diversity for Turkish P. theophrasti populations was found to be 18.60% and for Cretan populations 13.45%. Antalya–Kumluca–Karaöz samples were grouped outside the branches of the remaining P. theophrasti samples. All three Cretan populations formed their own, separate branch. Datça–Eksera Stream samples together with two Datça–Hurmalıbük specimens constituted a group excluding Datça–Hurmalıbük and Bodrum–Gölköy specimens. Five Bodrum–Gölköy genotypes were clustered separately. Gene flow (Nm) values among populations were estimated from 0.157 to 59.615. AMOVA analysis revealed the percentages of variance among and within Phoenix populations: 73% and 27%, respectively. The first three principal coordinate components accounted for 37.60, 29.32 and 20.04%, respectively, thus the total variance obtained from the first three principal coordinate components was 86.96%. A positive correlation between geographic and genetic distances of populations was detected by Mantel tests (Rx,y = 0.44, p = 0.04). The populations were classified into four clusters by STRUCTURE analysis, supported the PCoA data. To conclude, ISSR results support that P. dactylifera and P. theophrasti are different species. Moreover, the findings not only revealed relationships between natural Phoenix theophrasti populations but also supported the identification of the P. theophrasti individuals that are phenotypically differentiated in the divided Bodrum–Gölköy population (P. theophrasti subsp. golkoyana).This work was supported by the Scientific Research Projects Coordination Unit of Isik University, Project No. 16A401.Publisher's Versio

Cross-species hybridization and the origin of North African date palms

International audienceDate palm ( Phoenix dactylifera L.) is a major fruit crop of arid regions that were domesticated ∼7,000 y ago in the Near or Middle East. This species is cultivated widely in the Middle East and North Africa, and previous population genetic studies have shown genetic differentiation between these regions. We investigated the evolutionary history of P. dactylifera and its wild relatives by resequencing the genomes of date palm varieties and five of its closest relatives. Our results indicate that the North African population has mixed ancestry with components from Middle Eastern P. dactylifera and Phoenix theophrasti , a wild relative endemic to the Eastern Mediterranean. Introgressive hybridization is supported by tests of admixture, reduced subdivision between North African date palm and P. theophrasti , sharing of haplotypes in introgressed regions, and a population model that incorporates gene flow between these populations. Analysis of ancestry proportions indicates that as much as 18% of the genome of North African varieties can be traced to P. theophrasti and a large percentage of loci in this population are segregating for single-nucleotide polymorphisms (SNPs) that are fixed in P. theophrasti and absent from date palm in the Middle East. We present a survey of Phoenix remains in the archaeobotanical record which supports a late arrival of date palm to North Africa. Our results suggest that hybridization with P. theophrasti was of central importance in the diversification history of the cultivated date palm

A Common Approach to the Conservation of Threatened Island Vascular Plants: First Results in the Mediterranean Basin

The Mediterranean islands represent a center of vascular plant diversity featuring a high rate of endemic richness. Such richness is highly threatened, however, with many plants facing the risk of extinction and in need of urgent protection measures. The CARE-MEDIFLORA project promoted the use of ex situ collections to experiment with in situ active actions for threatened plants. Based on common criteria, a priority list of target plant species was elaborated, and germplasm conservation, curation and storage in seed banks was carried out. Accessions were duplicated in the seed banks of the partners or other institutions. Germination experiments were carried out on a selected group of threatened species. A total of 740 accessions from 429 vascular plants were stored in seed banks, and 410 seed germination experiments for 283 plants species were completed; a total of 63 in situ conservation actions were implemented, adopting different methodological protocols. For each conservation program, a specific monitoring protocol was implemented in collaboration with local and regional authorities. This project represents the first attempt to develop common strategies and an opportunity to join methods and methodologies focused on the conservation of threatened plants in unique natural laboratories such as the Mediterranean islands

An early evaluation of translocation actions for endangered plant species on Mediterranean islands

In situ conservation is widely considered a primary conservation strategy. Plant translocation, specifically, represents an important tool for reducing the extinction risk of threatened species. However, thus far, few documented translocations have been carried out in the Mediterranean islands. The Care-Mediflora project, carried out on six Mediterranean islands, tackles both short- and long-term needs for the insular endangered plants through in situ and ex situ conservation actions. The project approach is based on using ex situ activities as a tool to improve in situ conservation of threatened plant species. Fifty island plants (representing 45 taxa) were selected for translocations using common criteria. During the translocations, several approaches were used, which differed in site selection method, origin of genetic material, type of propagative material, planting method, and more. Although only preliminary data are available, some general lessons can be learned from the experience of the Care-Mediflora project. Among the factors restricting the implementation of translocations, limited financial resources appear to be the most important. Specific preliminary management actions, sometimes to be reiterated after translocation, increase the overall cost, but often are necessary for translocation success. Translocation using juvenile/reproductive plants produces better results over the short term, although seeds may provide good results over the long run (to be assessed in the future). Regardless, plant translocation success can only be detected over long periods; therefore, proper evaluation of plant translocations requires a long-term monitoring protocol. Care-Mediflora project represents the first attempt to combine the existing approaches in a common plant conservation strategy specifically focusing on the Mediterranean islands. Keywords: Care-Mediflora project, ex situ conservation, in situ conservation, Insular flora, Threatened plant population

Data from: Cross-species hybridization and the origin of North African date palms

Date palm (Phoenix dactylifera L.) is a major fruit crop of arid regions that were domesticated 7,000 y ago in the Near or Middle East. This species is cultivated widely in the Middle East and North Africa, and previous population genetic studies have shown genetic differentiation between these regions. We investigated the evolutionary history of P. dactylifera and its wild relatives by resequencing the genomes of date palm varieties and five of its closest relatives. Our results indicate that the North African population has mixed ancestry with components from Middle Eastern P. dactylifera and Phoenix theophrasti, a wild relative endemic to the Eastern Mediterranean. Introgressive hybridization is supported by tests of admixture, reduced subdivision between North African date palm and P. theophrasti, sharing of haplotypes in introgressed regions, and a population model that incorporates gene flow between these populations. Analysis of ancestry proportions indicates that as much as 18% of the genome of North African varieties can be traced to P. theophrasti and a large percentage of loci in this population are segregating for single-nucleotide polymorphisms (SNPs) that are fixed in P. theophrasti and absent from date palm in the Middle East. We present a survey of Phoenix remains in the archaeobotanical record which supports a late arrival of date palm to North Africa. Our results suggest that hybridization with P. theophrasti was of central importance in the diversification history of the cultivated date palm

SNPs from Flowers et al. (2019) in vcf format

This file contains SNPs used in Flowers et al. (2019) Cross-species hybridization and the origin of North African date palms in vcf format. Only SNPs passing filters are included in the file